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Huntington's disease (HD) is characterized by the dysfunction of mitochondrial energy metabolism, which is associated with the functional impairment of succinate dehydrogenase (mitochondrial complex II), and pyruvate dehydrogenase (PDH). Treatment with 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of succinate dehydrogenase, replicates most of the pathophysiological features of HD. In the present study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on 3-NP-induced cell injury in rat differentiated neuronal PC12 cells. The 3-NP caused cell necrosis, as assessed by lactate dehydrogenase (LDH) leakage, and mitochondrion-dependent cell apoptosis, as assessed by caspase-3 and caspase-9 activation, in differentiated PC12 cells. The cytotoxicity induced by 3-NP was associated with a depletion of cellular reduced glutathione (GSH) as well as the activation of redox-sensitive c-Jun N-terminal kinase (JNK) pathway and the inhibition of PDH. (-)Sch B pretreatment (5 and 15 μM) significantly reduced the extent of necrotic and apoptotic cell death in 3-NP-challenged cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with the attenuation of 3-NP-induced GSH depletion as well as JNK activation and PDH inhibition. (-)Sch B pretreatment enhanced cellular glutathione redox status and ameliorated the 3-NP-induced cellular energy crisis, presumably by suppressing the activated JNK-mediated PDH inhibition, thereby protecting against necrotic and apoptotic cell death in differentiated PC12 cells., (Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.)

We previously reported that hydrogen sulfide (H(2)S) produces protection in PC12 cells during 1-methy-4-phenylpyridinium ion (MPP(+)) challenge. The present study aims to clarify the mechanisms underlying the neuroprotective effects of H(2)S. We showed that both glybenclamide, an ATP-sensitive potassium (K(ATP)) channel blocker, and LY294002, a specific PI(3)K-AKT pathway inhibitor, reversed the neuroprotective effect of NaHS (a H(2)S donor) against MPP(+)-induced cytotoxicity to PC12 cells and that NaHS up-regulated the activity of AKT in PC12 cells, which was abolished by blockade of K(ATP) channels with glybenclamide. In addition, NaHS up-regulated the expression of Bcl-2 and blocked MPP(+)-induced down-regulation of Bcl-2, and this augmentation of Bcl-2 expression was prevented by both glybenclamide and LY294002. These data provided the evidence that the neuroprotective action of H(2)S against MPP(+) toxicity to PC12 cells is via the K(ATP)/PI(3)K/AKT/Bcl-2 pathway. We also demonstrated that NaHS attenuated the inhibitory effect of MPP(+) ERK1/2 activation in PC12 cells, whereas U0126, a specific MEK inhibitor, did not reverse the neuroprotective effect of NaHS, which indicated that attenuating MPP(+)-triggered down-regulation of ERK1/2 activation is involved in the protection of H(2)S against MPP(+) neurotoxicity, but ERK1/2 is not an essential effector mediating the neuroprotective effect of H(2)S. In conclusion, the present observations identify a crucial role of the K(ATP)/PI(3)K/AKT/Bcl-2 pathway in H(2)S-exerted neuroprotection against the toxicity of MPP(+). Findings from the present study will help shed light on the mechanisms of H(2)S-elicited neuroprotective effects on MPP(+) toxicity.

PC12 cells exhibit precise temporal control of growth factor signaling in which stimulation with epidermal growth factor (EGF) leads to transient extracellular signal-regulated kinase (ERK) activity and cell proliferation, whereas nerve growth factor (NGF) stimulation leads to sustained ERK activity and differentiation. While cyclic AMP (cAMP)-mediated signaling has been shown to be important in conferring the sustained ERK activity achieved by NGF, little is known about the regulation of cAMP and cAMP-dependent protein kinase (PKA) in these cells. Using fluorescence resonance energy transfer (FRET)-based biosensors localized to discrete subcellular locations, we showed that both NGF and EGF potently activate PKA at the plasma membrane, although they generate temporally distinct activity patterns. We further show that both stimuli fail to induce cytosolic PKA activity and identify phosphodiesterase 3 (PDE3) as a critical regulator in maintaining this spatial compartmentalization. Importantly, inhibition of PDE3, and thus perturbation of the spatiotemporal regulation of PKA activity, dramatically increases the duration of EGF-stimulated nuclear ERK activity in a PKA-dependent manner. Together, these findings identify EGF and NGF as potent activators of PKA activity specifically at the plasma membrane and reveal a novel regulatory mechanism contributing to the growth factor signaling specificity achieved by NGF and EGF in PC12 cells.

There is an intense discussion about the subcellular origin of the generation of reactive oxygen species (ROS) under hypoxia. Since this fundamental question can be addressed only in a cellular system, the O(2) -sensing rat pheochromocytoma (PC12) cells were used. Severe hypoxia is known to elevate non-esterified fatty acids. Therefore, the site(s) of ROS generation were studied in cells which we simultaneously exposed to hypoxia (1% oxygen) and free fatty acids (FFA). We obtained the following results: (i) at hypoxia, ROS generation increases in PC12 cells but not in mitochondria isolated therefrom. (ii) Non-esterified polyunsaturated fatty acids (PUFA) enhance the ROS release from PC12 cells as well as from mitochondria, both in normoxia and in hypoxia. (iii) PUFA-induced ROS generation by PC12 cells is not decreased either by inhibitors of the cell membrane NAD(P)H oxidase or inhibitors impairing the PUFA metabolism. (iv) PUFA-induced ROS generation of mitochondria is paralleled by a decline of the NADH-cytochrome c reductase activity (reflecting combined enzymatic activity of complex I plus III). (v) Mitochondrial superoxide indicator (MitoSOXred)-loaded cells exposed to PUFA exhibit increased fluorescence indicating mitochondrial ROS generation. In conclusion, elevated PUFA levels enhance cellular ROS level in hypoxia, most likely by impairing the electron flux within the respiratory chain. Thus, we propose that PUFAs are likely to act as important extrinsic factor to enhance the mitochondria-associated intracellular ROS signaling in hypoxia., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Sixteen new neolignans and lignans (1-16), together with 12 known analogues, have been isolated from an ethanol extract of the bark of Machilus robusta. Compounds 1 and 2 showed activity against HIV-1 replication in vitro, with IC(50) values of 2.52 and 2.01 μM, respectively. At 10 μM, 6, 8, and 9 reduced dl-galactosamine-induced hepatocyte (WB-F344 cells) damage, and 9 could additionally attenuate rotenone-induced PC12 cell damage. The known compounds (-)-pinoresinol (17) and (+)-lyoniresinol (18) were active against serum deprivation induced PC12 cell damage.

Background: Kainic acid (KA)-induced status epilepticus (SE) was involved with release of free radicals. Sesamin is a well-known antioxidant from sesame seeds and it scavenges free radicals in several brain injury models. However the neuroprotective mechanism of sesamin to KA-induced seizure has not been studied., Methods: Rodents (male FVB mice and Sprague-Dawley rats) were fed with sesamin extract (90% of sesamin and 10% sesamolin), 15 mg/kg or 30 mg/kg, for 3 days before KA subcutaneous injection. The effect of sesamin on KA-induced cell injury was also investigated on several cellular pathways including neuronal plasticity (RhoA), neurodegeneration (Caspase-3), and inflammation (COX-2) in PC12 cells and microglial BV-2 cells., Results: Treatment with sesamin extract (30 mg/kg) significantly increased plasma α-tocopherol level 50% and 55.8% from rats without and with KA treatment, respectively. It also decreased malondialdehyde (MDA) from 145% to 117% (p=0.017) and preserved superoxide dismutase from 55% of the vehicle control mice to 81% of sesamin-treated mice, respectively to the normal levels (p=0.013). The treatment significantly decreased the mortality from 22% to 0% in rats. Sesamin was effective to protect PC12 cells and BV-2 cells from KA-injury in a dose-dependent manner. It decreased the release of Ca2+, reactive oxygen species, and MDA from PC12 cells. Western blot analysis revealed that sesamin significantly reduced ERK1/2, p38 mitogen-activated protein kinases, Caspase-3, and COX-2 expression in both cells and RhoA expression in BV-2 cells. Furthermore, Sesamin was able to reduce PGE2 production from both cells under KA-stimulation., Conclusions: Taken together, it suggests that sesamin could protect KA-induced brain injury through anti-inflammatory and partially antioxidative mechanisms.

Aims: We investigated the mechanism of D-galactose (DG)-induced oxidative damage and the neuroprotective action of genistein in PC12 cells., Main Methods: PC12 cells were treated with 40mM DG dissolved in medium containing 85% RPMI1640, 10% HBS and 5% FBS with or without genistein. We measured the protein expression of β-amyloid (Aβ), advanced glycation end products (AGEs), IκB-α and manganese-superoxide dismutase (MnSOD) by western blotting, intracellular reactive oxygen species (ROS) by 2, 7-dichlorofluorescin-diacetate, and the binding activity of nuclear factor kappa B (NF-κB) by electrophortic mobility shift assay., Key Findings: DG (40mM) completely retarded cell growth after incubation for 72h, and this effect was not due to osmotic changes, as 40mM mannitol had no effect. Mechanistically, we found that DG increased intracellular ROS starting at 4h and increased Aβ and AGEs at 24h. DG treatment for 24h also increased the binding activity of NF-κB but strongly decreased the expression of IκB-α protein. Furthermore, DG treatment for 48h increased MnSOD protein expression. All these effects of DG were effectively inhibited by genistein (0.5-10μM)., Significance: The present study indicates that the protection of genistein against DG-induced oxidative stress in PC12 cells, and the effect is likely mediated by decreased intracellular ROS and binding activity of NF-κB., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Insulin-like growth factor 1 (IGF-1) stimulates α-secretase processing of amyloid precursor protein (APP) and decreases Aβ production. Little is known about the relationship between IGF-1 and β-site amyloid precursor protein cleaving enzyme 1 (BACE-1), the protease essential for the production of β-amyloid peptides (Aβ). Here, we investigated the effect of IGF-1 on BACE-1 in PC12 cells. Quantitative polymerase chain reaction analysis and western blot showed that treatment of cells with IGF-1 significantly decreased the levels of BACE-1 mRNA and protein. Furthermore, IGF-1 increased the phosphorylation of Akt and ERK1/2. The presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 and the mitogen-activated protein kinase kinases (MEK) inhibitor PD98059 blocked the effect of IGF-1 on BACE-1. Our data indicated that IGF-1-induced reduction of BACE-1 might involve the PI3-K/Akt and MAPK/ERK1/2 signaling pathways.

The in vitro labeling of stem or therapeutic cells with engineered nanoparticles with the aim of transplanting these cells into live animals and, for example, noninvasively monitoring their migration, is a hot topic in nanomedicine research. It is of crucial importance that cell-nanoparticle interactions are studied in depth in order to exclude any negative effects of the cell labeling procedure. To date, many disparate results can be found in the literature regarding nanoparticle toxicity due to the great versatility of different parameters investigated. In the present work, an overview is presented of different types of nanomaterials, focusing mostly on iron oxide nanoparticles, developed for biomedical research. The difficulties in assessing nanoparticle-mediated toxicity are discussed, an overview of some of the problems encountered using commercial (dextran-coated) iron oxide nanoparticles is presented, several key parameters are highlighted and novel methods suggested--emphasizing the importance of intracellular nanoparticle degradation and linking toxicity data to functional (i.e., cell-associated) nanoparticle levels, which could help to advance any progress in this highly important research topic.

The rat pheochromocytoma cell line PC12 has been widely used as a model to study neuronal differentiation. PC12 cells give rise to neurites in response to basic fibroblast growth factor (bFGF). However, it is unclear whether bFGF promotes neurite outgrowth by inducing RhoA inactivation, and a mechanism for RhoA inactivation in PC12 cells in response to bFGF has not been reported. Lysophosphatidic acid (LPA) treatment and the expression of constitutively active (CA)-RhoA (RhoA V14) impaired neurite formation in response to bFGF, while Tat-C3 exoenzyme and the expression of dominant negative (DN)-RhoA (RhoA N19) stimulated neurite outgrowth. GTP-bound RhoA levels were reduced in response to bFGF, which suggests that the inactivation of RhoA is essential to neurite outgrowth in response to bFGF. To investigate the mechanism of RhoA inactivation, this study examined the roles of p190RhoGAP and Rap-dependent RhoGAP (ARAP3). DN-p190RhoGAP prevented neurite outgrowth, while WT-p190RhoGAP and Src synergistically stimulated neurite outgrowth; these findings suggest that bFGF promotes the inactivation of RhoA and subsequent neurite outgrowth through p190RhoGAP and Src. Furthermore, DN-Rap1 and DN-ARAP3 reduced neurite formation in PC12 cells. These results suggest that RhoA is likely to be inactivated by p190RhoGAP and ARAP3 during neurite outgrowth in response to bFGF., ((c) 2010 Wiley-Liss, Inc.)

Proteasomal dysfunction may play a role in neurodegenerative conditions and protein aggregation. Overexpression in neuronal cells of alpha-synuclein, a molecule linked to Parkinson's Disease, may lead to proteasomal dysfunction. Using PC12 cells stably expressing wild-type or mutant alpha-synuclein and gel filtration, we demonstrate that soluble, intermediate size oligomers of alpha-synuclein co-elute with the 26S proteasome. These soluble oligomers associate with the 26S proteasome and are significantly increased following treatment with proteasomal, but not lysosomal, inhibitors, indicating specific degradation of these particular species by the 26S proteasome. Importantly, expression of alpha-synuclein resulted in a significant inhibition of all proteasomal activities without affecting the levels or assembly of the 26S proteasome. Pharmacological dissociation of alpha-synuclein oligomers restored proteasomal function and reduced polyubiquitinated protein load in intact cells. Our findings suggest a model where only a subset of specific soluble cell-derived alpha-synuclein oligomers is targeted to the 26S proteasome for degradation, and simultaneously inhibit its function, likely by impeding access of other proteasomal substrates., (Copyright 2008 Elsevier Inc. All rights reserved.)

Endothelial cells (ECs) from different vascular beds not only display common characteristics but are also quite heterogeneous in terms of expression and secretion of neuro-angiogenic factors, which may help explain some of their distinct physiological roles. We investigated by RT-PCR the gene expression, by PC12 bioassay the neurotropic activity, and by ELISAs the levels of NGF and FGF-2 using conditioned medium collected from cultures of ECs derived from myocardial and cerebral capillaries. While NGF was expressed and released by both cell types, FGF-2 was expressed and released solely by the brain but not heart ECs. Oxygen-glucose deprivation (ischemic) insult blocked NGF secretion from heart and brain ECs and inhibited by 70% the secretion of FGF-2 from brain ECs. We propose that the differential expression of NGF and FGF-2 in heart and brain EC cultures reflect heterogeneity on demand of the microcapillary components and the surrounding microenvironment for a proper tissue-specific homeostasis.

Background: Brominated flame retardants, including the widely used polybrominated diphenyl ethers (PBDEs), have been detected in humans, raising concern about possible neurotoxicity. Recent research demonstrated that the hydroxylated metabolite 6-OH-BDE-47 increases neurotransmitter release by releasing calcium ions (Ca2+) from intracellular stores at much lower concentrations than its environmentally relevant parent congener BDE-47. Recently, several other hydroxylated BDE-47 metabolites, besides 6-OH-BDE-47, have been detected in human serum and cord blood., Objective and Methods: To investigate the neurotoxic potential of other environmentally relevant PBDEs and their metabolites, we examined and compared the acute effects of BDE-47, BDE-49, BDE-99, BDE-100, BDE-153, and several metabolites of BDE-47-6-OH-BDE-47 (and its methoxylated analog 6-MeO-BDE-47), 6 -OH-BDE-49, 5-OH-BDE-47, 3-OH-BDE-47, and 4 -OH-BDE-49--on intracellular Ca2+ concentration ([Ca2+]i), measured using the Ca2+-responsive dye Fura-2 in neuroendocrine pheochromocytoma (PC12) cells., Results: In contrast to the parent PBDEs and 6-MeO-BDE-47, all hydroxylated metabolites induced Ca2+ release from intracellular stores, although with different lowest observed effect concentrations (LOECs). The major intracellular Ca2+ sources were either endoplasmic reticulum (ER; 5-OH-BDE-47 and 6 -OH-BDE-49) or both ER and mitochondria (6-OH-BDE-47, 3-OH-BDE-47, and 4 -OH-BDE-49). When investigating fluctuations in [Ca2+]i, which is a more subtle end point, we observed lower LOECs for 6-OH-BDE-47 and 4 -OH-BDE-49, as well as for BDE-47., Conclusions: The present findings demonstrate that hydroxylated metabolites of BDE-47 cause disturbance of the [Ca2+]i. Importantly, shielding of the OH group on both sides with bromine atoms and/or the ether bond to the other phenyl ring lowers the potency of hydroxylated PBDE metabolites.

In vitro studies indicated that hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have an increased toxic potential compared to their parent congeners. An example is the OH-PBDE-induced increase of basal intracellular Ca(2+) concentration ([Ca(2+)](i)) by release of Ca(2+) from endoplasmic reticulum (ER) and mitochondria and/or influx of extracellular Ca(2+). ER and mitochondria regulate Ca(2+) homeostasis in close association with voltage-gated Ca(2+) channels (VGCCs). Therefore, effects of (OH-)PBDEs on the depolarization-evoked (100 mM K(+)) net increase in [Ca(2+)](i) (depolarization-evoked [Ca(2+)](i)) were measured in neuroendocrine pheochromocytoma cells using the Ca(2+)-responsive dye Fura-2. OH-PBDEs dose dependently inhibited depolarization-evoked [Ca(2+)](i). This inhibition was potentiated by a preceding increase in basal [Ca(2+)](i). Especially at higher concentrations of OH-PBDEs (5-20 microM), large increases in basal [Ca(2+)](i) strongly inhibited depolarization-evoked [Ca(2+)](i). The inhibition appeared more sensitive to increases in basal [Ca(2+)](i) by Ca(2+) release from intracellular stores (by 3-OH-BDE-47 or 6'-OH-BDE-49) compared to those by influx of extracellular Ca(2+) (by 6-OH-BDE-47 or 5-OH-BDE-47). The expected [Ca(2+)](i) difference close to the membrane suggests involvement of Ca(2+)-dependent regulatory processes close to VGCCs. When coapplied with depolarization, some OH-PBDEs induced also moderate direct inhibition of depolarization-evoked [Ca(2+)](i). Polybrominated diphenyl ethers and methoxylated BDE-47 affected neither basal nor depolarization-evoked [Ca(2+)](i), except for BDE-47, which moderately increased fluctuations in basal [Ca(2+)](i) and depolarization-evoked [Ca(2+)](i). These findings demonstrate that OH-PBDEs inhibit depolarization-evoked [Ca(2+)](i) depending on preceding basal [Ca(2+)](i). Related environmental pollutants that affect Ca(2+) homeostasis (e.g., polychlorinated biphenyls) may thus also inhibit depolarization-evoked [Ca(2+)](i), justifying further investigation of possible mixture effects of environmental pollutants on Ca(2+) homeostasis.

Diabetic neuropathy is one of the most frequent complications of diabetes. Despite some studies, the exact mechanism of glucose neurotoxicity has not been fully elucidated. Increased reactive oxygen species (ROS) has proposed as a possible mechanism. Crocus sativus L. (saffron) has been known as a source of antioxidants. Therefore, neuroprotective effect of saffron extract, its active component crocin and gamma-glutamylcysteinylglycine (GSH) was studied in glucose-induced neurotoxicity, using PC12 cells as a suitable in vitro model of diabetic neuropathy. Cell viability was quantitated by MTT assay. ROS was measured using DCF-DA by flow cytometry analysis. The result showed that glucose (13.5 and 27 mg/ml) reduced the cell viability of PC12 cells after 4 days. Saffron extract (5 and 25 mg/ml), crocin (10 and 50 muM) and GSH (10 muM) could decrease this toxicity. Glucose toxicity was consistent with increased ROS production which reduced by saffron, crocin and GSH pretreatment. These results suggest saffron and its carotenoid crocin could be potentially useful in diabetic neuropathy treatment.

Cell-based approaches are a promising therapeutic strategy for treating injuries to the nervous system, but the optimal means to promote neurite extension and direct cellular behavior are unclear. Previous studies have examined the behavior of neural-like cells in ambient air (21% oxygen tension), yet these conditions are not representative of the physiological oxygen microenvironment of neural tissues. We hypothesized that neuronal differentiation of a model neural cell line (PC12) could be controlled by modulating local oxygen tension. Compared to ambient conditions, PC12 cells cultured in reduced oxygen exhibited significant increases in neurite extension and total neurite length, with 4% oxygen yielding the highest levels of both indicators. We confirmed neurite extension was mediated through oxygen-responsive mechanisms using small molecules that promote or inhibit HIF-1alpha stabilization. The hypoxic target gene Vegf was implicated as a neurotrophic factor, as neurite formation at 21% oxygen was mimicked with exogenous VEGF, and a VEGF-neutralizing antibody attenuated neurite formation under reduced oxygen conditions. These findings demonstrate that behavior of neural-like cells is driven by the oxygen microenvironment via VEGF function, and suggest promising approaches for future applications in neural repair.

Oxidative stress plays an important role in many neurodegenerative disorders. In this study, the effect of baicalein, a natural flavonoid isolated from the root of Scutellaria baicalensis G., on hydrogen peroxide (H2O2)-induced cytotoxicity in PC12 cells were investigated. Exposure of PC12 cells to 0.15 mM H2O2 for 20 min induced a significant decrease in cell viability accompanied by increased oxidative stress, mitochondrial dysfunction, downregulation of Bcl-2, upregulation of Bax, and cell apoptosis. Pretreatment of PC12 cells with baicalein inhibited H2O2-induced cell viability loss, intracellular reactive oxygen species generation, and lipid peroxidation in a dose-dependent manner. Meanwhile, baicalein potentially inhibited H2O2-induced cell apoptosis characterized with the DNA fragment. And the mitochondrial pathway involving the mitochondrial dysfunction associated with cell apoptosis including membrane potential loss, the release of cytochrome c, the downregulation of Bcl-2, upregulation of Bax induced by H2O2 were also abrogated in the presence of baicalein. Taken together, these results suggest that baicalein can block H2O2-induced apoptosis by prevention of oxidative stress as well as regulation of Bcl-2 family members and suppression of mitochondria dysfunction, which might be beneficial for the treatment of oxidative stress in aging and age-associated neurodegenerative diseases.

n-Hexanal (hexanal), a straight-chain six-carbon aldehyde, is mainly present in plants. Hexanal strongly affects the release of dopamine from rat striatal slices and rat pheochromocytoma (PC12) cells. In this study, we attempted to clarify the mechanism underlying the regulation of dopamine release by hexanal by using PC12 cells, which have the ability to synthesize, store, and release dopamine. The stimulation of PC12 cells with hexanal enhanced dopamine release in a time- and dose-dependent manner. Dopamine release was partially inhibited by pretreatment of the cells with BAPTA-AM, a cell-permeable Ca2+ chelator. In addition, the intracellular Ca2+ concentration was found to slowly increase after hexanal stimulation. Furthermore, the Src tyrosine kinase inhibitor PP2 partially inhibited hexanal-induced dopamine release. However, the levels of phosphorylated proteins decreased after hexanal stimulation. Hexanal stimulated the release of only a small amount of dopamine from reserpine-treated PC12 cells, in which the vesicular dopamine was depleted. These findings suggest that both an increase in the intracellular Ca2+ concentration and the dephosphorylation of phosphorylated proteins might be required for hexanal-stimulated release of dopamine, and that the dopamine released because of hexanal stimulation mainly comes from the dopamine vesicles. This study showed the cellular events that occurred in PC12 cells after stimulation of hexanal. Furthermore, it is important to examine the relationship between the cellular functions and the physiological effects of hexanal on dopamine release.

The secretion of the hormone insulin from beta cells is modulated by the expression of the dense core vesicle transmembrane protein IA-2. Since IA-2 is found in neuroendocrine cells throughout the body, the present experiments were initiated to determine whether the expression of IA-2 also modulates the secretion of neurotransmitters. Using the dopamine-secreting pheochromocytoma cell line PC12, we found that the overexpressions of IA-2 increased the cellular content and secretion of dopamine, whereas the knockdown of IA-2 by siRNA decreased the cellular content and secretion of dopamine. Neither the overexpression nor knockdown of IA-2 influenced the uptake of [H(3)]dopamine by PC12 cells, but did influence the amount of [H(3)]dopamine secreted. Overexpression of IA-2 also increased the level of the dense core vesicle-associated proteins Rab3A, IA-2beta and secretogranin II, whereas the knockdown of IA-2 decreased the level of these proteins. We conclude that the expression of IA-2 profoundly influences the function of dense core vesicles and has a broad modulating effect on the cellular content and secretion of both hormones and neurotransmitters., (Published by Elsevier Ireland Ltd.)

Expression of the nuclear orphan receptor gene Nur77 in neuronal cells is induced by activity-dependent increases in intracellular Ca2+ ions. Ca2+ responsiveness of the Nur77 gene has been attributed to two distinct DNA regulatory regions that recruit the transcription factors cAMP response element binding protein (CREB) and myocyte enhancer factor-2 (MEF2). Here we used dominant interfering and constitutively active mutants of CREB and MEF2 proteins to assess their relative contribution to depolarization-induced Nur77 expression in undifferentiated PC12 cells and hippocampal neurons. We show that while CREB is necessary for Ca2+-activated Nur77 expression MEF2 functions to modulate CREB-dependent Nur77 expression by acting as a repressor in quiescent cells.

Oxidative damage is believed to be associated with ageing, cancer and several degenerative diseases. Previous reports have shown that safflower-seed extract and its major antioxidant constituents, serotonin hydroxycinnamic amides, possess a powerful free radical-scavenging and antioxidative activity, paying particular attention to atherosclerotic reactive oxygen species (ROS)-related dysfunctions. In the present report, we examined a still unknown cell-based mechanism of serotonin derivatives against ROS-related neuronal damage, phenomena that represent a crucial event in neurodegenerative diseases. Serotonin derivatives N-(p-coumaroyl)serotonin and N-feruloylserotonin exerted a protective effect on high glucose-induced cell death, inhibited the activation of caspase-3 which represents the last and crucial step within the cascade of events leading to apoptosis, and inhibited the overproduction of the mitochondrial superoxide, which represents the most dangerous radical produced by hyperglycaemia, by acting as scavengers of the superoxide radical. In addition, serotonin derivative concentration inside the cells and inside the mitochondria was increased in a time-dependent manner. Since recent studies support the assertion that mitochondrial dysfunctions related to oxidative damage are the major contributors to neurodegenerative diseases, these preliminary cell-based results identify a mitochondria-targeted antioxidant property of serotonin derivatives that could represent a novel therapeutic approach against the neuronal disorders and complications related to ROS.

Background: Merkel cells (MCs) exist in the epidermal basal layer, in contact with keratinocytes. This direct contact seems critical for maintaining MCs in vitro., Objectives: To estimate the effects of nerve cells on the maintenance of MCs within epidermal sheets in a new organotypic culture system of MCs., Methods: We developed a new organotypic culture system of MCs, using MC-containing epidermal sheets embedded in collagen gel. To estimate the effects of nerve cells on the maintenance of MCs within the epidermal sheets, we cocultured nerve cells and MC-containing epidermal sheets. In these culture assemblies, cellular behaviour was analysed by histochemistry, immunohistochemistry, electron microscopy and enzyme-linked immunosorbent assay., Results: This culture, even in the absence of neurotrophin (NT)-3 and nerve growth factor (NGF) (which are crucial for MC biology), retained cytokeratin (CK)-20-positive and neuroendocrine granule-containing MCs within the sheets for over 2 weeks. Coculture of MCs with PC-12 nerve cells significantly increased the number of MCs within the epidermal sheets, and the keratinocytes had almost identical expression levels of CK1, CK10, CK14 and the progenitor marker p63 to those produced by keratinocytes in vivo. Uptake of the growth marker bromodeoxyuridine by MCs and levels of NT-3 and NGF in the culture supernatants were undetectable in this system, regardless of the presence or absence of PC-12., Conclusions: The data suggest, first, that direct contact between MCs and keratinocytes may be critical for retaining MCs in vitro; second, that nerve cell-affected maintenance of keratinocyte differentiation, but not NT-3 and NGF, may contribute to MC maintenance; and third, that MCs are not able to grow, at least in our system. Our method would be useful for studying MC biology.

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

Lipid droplets are cytoplasmic organelles found in almost all cells under physiological or pathological conditions. Certain nanoparticles can induce lipid droplet formation under oxidative stress conditions. Small metallic nanoparticles such as cadmium telluride (CdTe) nanoparticles, particularly those with incompletely protected surfaces, induce oxidative stress and may inflict damages to several intracellular organelles. The objective of this study was to assess formation of lipid droplets in cells treated with CdTe nanoparticles and relate their status to cell function (mitochondrial activity and cell viability). Multicolor labeling of cellular organelles (lipid droplets and lysosomes) showed that lipid droplets formed in pheochromocytoma (PC12) cells following nanoparticle or oleic acid treatment. Some lipid droplets were found closely apposed to lysosomes suggesting possible communication between these organelles during severe oxidative stress. Combination of microscopy of living cells with cell viability assays showed that oleic acid-induced lipid droplets not only serve as intracellular lipid storage sites but also play a protective role in starving stressed cells. Results from these studies suggest that oleic acid-induced LD in PC12 cells are dynamic and adaptive organelles, which provide energy to starving cells and facilitate their rescue under starvation and exposure to metallic nanoparticles.

Herein we demonstrate that PC12 cells, which overexpress human wild-type amyloid-beta precursor protein (AbetaPPwt) or AbetaPP bearing double Swedish mutation (AbetaPPsw), reveal phenotype characteristic for Alzheimer's disease (AD). The examination of cell ultrastructure showed the presence of peptide aggregates within the cells, activation of endosomal-lysosomal system and extensive exocytosis. Furthermore, the autophagy induction was also characteristic hallmark of amyloid-beta-induced cytotoxicity. Morphological changes were positively correlated with the extent of phosphorylated glycogen synthase kinase-3beta (phospho-Tyr(216)-GSK-3beta, GSK-3beta-P(Y216)). The activity of GSK-3beta is believed to cause tau protein hyper-phosphorylation, increased amyloid-beta production and local plaque-associated microglial-mediated inflammatory responses. All of them are symptomatic for AD. In our studies, the highly significant Y216 phosphorylation and over-expression of total GSK-3beta were observed in AbetaPPsw-transfected PC12 cells. In addition, the immuocytochemical analysis showed co-localization of GSK-3beta-P(Y216) and amyloid-beta deposits. Thus, our data support a functional role of GSK-3beta in AbetaPP processing, further implicating this kinase in the amyloid-beta-dependent pathogenesis.

Protein recruitment during endocytosis is well characterized in fibroblasts. Since fibroblasts do not engage in regulated exocytosis, only information about protein recruitment during constitutive endocytosis is provided. Furthermore, the cortical actin of fibroblasts is characterized by stress fibers rather than a thick cortical meshwork. A cell model, which differs in these features, could provide insight into the heterogeneity of protein recruitment to constitutive and exocytosis coupled endocytotic areas. Therefore, this study investigates the sequence of protein recruitment in PC12 cells, a well documented exocytotic cell model with thick actin cortex. Using real time total-internal-reflection fluorescence microscopy it was found that at the plasma membrane steady, but not transient, dynamin-1-EGFP or -mCherry fluorescence spots that rapidly dimmed coincided with markers for constitutive endocytotic such as clathrin-LC-dsRed and transferrin-receptor-pHluorin. Clathrin-LC-dsRed and dynamin-1-EGFP were further used to determine the temporal sequence of protein recruitment to areas of constitutive endocytosis. mCherry- and EGFP-beta-actin, Arp-3-EGFP and EGFP-mAbp1 were slowly recruited before the dynamin-1-mCherry fluorescence dimmed, but their fluorescence peaked after the loss of clathrin-LC-dsRed commenced. Furthermore, mCherry-beta-actin fluorescence increased before exocytosis, indicating redistribution prior to release. Also, no average dynamin-1-mCherry recruitment was observed within 50 s to regions of exocytosis marked by NPY-mGFP. This indicates that the temporal-spatial coupling between regulated exo-and endocytosis is rather limited in PC12 cells. Furthermore, the time course of the protein recruitment to constitutive endocytotic sites might depend on the subcellular morphology such as the size of the actin cortex.

The assembly of amyloid beta-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid beta-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid beta-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.

Background: Pheochromocytomas are rare tumors derived from the chromaffin cells of the adrenal medulla. Although these tumors have long been postulated to induce hypertension and cardiomyopathy through the hypersecretion of catecholamines, catecholamines alone may not fully explain the profound myocardial remodeling induced by these tumors. We sought to determine whether changes in myocardial function in pheochromocytoma-induced cardiomyopathy result solely from catecholamines secretion or from multiple pheochromocytoma-derived factors., Methods and Results: Isolated cardiomyocytes incubated with pheochromocytoma-conditioned growth media contracted at a higher frequency than cardiomyocytes incubated with norepinephrine (NE) only. Sprague-Dawley rats and black-6 mice were implanted with agarose-encapsulated pheochromocytoma (PC12) cells, dihydroxyphenylalanine decarboxylase knock-out PC12 cells deficient in NE (PC12-KO), or NE-secreting pumps. PC12 cell implantation increased left ventricular dilation by 35+/-6% and 9.6+/-1.4% and reduced left ventricular fractional shortening by 20+/-3% and 28+/-4% in rats and mice compared with animals dosed only with NE, respectively. Elimination of NE secretion in PC12-KO cells induced neither cardiac dilation (3.9%+/-1.8% increase versus control) nor changes in (1.9%+/-0.4% reduction) fractional shortening compared to controls., Conclusions: Pheochromocytomas induce a greater degree of cardiomyopathy than equivalent doses of NE, suggesting pheochromocytoma-induced cardiomyopathy is not solely mediated by NE, rather pheochromocytoma secretory factors in combination with catecholamines act synergistically to induce greater cardiac damage than catecholamines alone.

Optimum time points for oxygen-glucose deprivation (OGD) and re-oxygenation have been identified to suggest the suitability of PC-12 cells as rapid and sensitive in vitro model of cerebral stroke. Further, the precise role of glucose as one of the limiting factors was ascertained. PC-12 cells were subjected to receive OGD of 1-8 h followed by re-oxygenation for 6 to 96 h in medium having glucose 0-10 mg/ml. Loss of cell viability was assessed using trypan blue dye exclusion and MTT assays. The significant (p < 0.05) reduction in percent viable cell count was started at 2 h of OGD (80.7 +/- 2.0) and continued in further OGD periods (3, 4, 5, 6, 7, and 8 h), i.e. 65.7 +/- 3.5, 59.7 +/- 4.6, 54.3 +/- 3.2, 44.7 +/- 2.9, 20.3 +/- 4.3, 5.7 +/- 2.0 of counted cells, respectively. Cells growing in glucose-free medium have shown a gradual (p < 0.001) decrease in cell viability throughout the re-oxygenation. Re-oxygenation of 24 h was found to be first statistically significant time point for all the glucose concentrations. Glucose concentration during re-oxygenation was found to be one of the key factors involved in the growth and proliferation in PC-12 cells. The OGD of 6 h followed by a re-oxygenation period of 24 h with 4-6 mg/ml glucose concentration could be recorded as optimum conditions under our experimental conditions.

Mammalian cells require a constant O2 supply to produce adequate energy, and sustained hypoxia can kill cells. Mammals therefore have evolved sophisticated mechanisms to allow their cells to adapt to hypoxia. In this study, we investigated the role of TRP channels and the Na+-Ca2+ exchanger (NCX) in mediating hypoxia-induced [Ca2+]i elevation in a model of the O2-sensing rat pheochromocytoma (PC12) cell line by using Ca2+ imaging and molecular biological approaches. Non-selective cation channels, such as TRPC1, 3 and 6, were found to be functionally expressed in PC12 cells. They mediated Ca2+ entry when cells were exposed to acute hypoxia (PO2 of 15 mmHg), in addition to Ca2+ entry via VGCCs. Blockage of TRPCs by 2APB and SKF96365 could significantly reduce hypoxia-mediated [Ca2+]i elevation. Suramin and U73122 attenuated the hypoxia-induced [Ca2+]i elevation, implying the involvement of the G-protein and PLC pathways in the hypoxic response. In addition to TRPCs and VGCCs, NCX also contributed to the hypoxia-induced [Ca2+]i elevation, and blockade of NCX by KBR7943 could significantly decrease the hypoxia-induced [Ca2+]i elevation. Our results suggest that the activation of TRP by hypoxia could lead to NCX reversal; furthermore, membrane depolarization and TRPCs may play a primary role in mediating the hypoxic response in PC12 cells.

Background: The histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) have been demonstrated recently to be strong Notch-1 activators. Upregulation of the Notch-1 pathway has been shown to limit growth and suppress hormonal secretion in neuroendocrine (NE) neoplasms. We hypothesized that HDAC inhibition would be an effective strategy to activate the Notch-1 pathway and inhibit growth and hormonal secretion in pheochromocytoma cells., Methods: Pheochromocytoma PC-12 cells were treated with up to 8 mmol/L VPA or 40 micromol/L SBHA for 2 days. NE tumor markers achaete-scute complex-like 1 (ASCL1) and chromogranin A (CgA) were measured by Western analysis after treatment. Growth was assessed by a cellular proliferation assay; Western analysis was used to determine the mechanism of growth regulation., Results: HDAC inhibitor treatment caused a dose-dependent decrease in ASCL1 and CgA while increasing the amount of active Notch-1 protein; with a 6-day treatment, dose-dependent growth inhibition and cleavage of the apoptotic markers caspase-3 and poly-ADP ribose phosphate was observed., Conclusion: VPA and SBHA upregulate Notch-1 effectively, suppress NE tumor markers, and decrease growth via apoptosis of pheochromocytoma cells in vitro. Activation of the Notch-1 signaling pathway with HDAC inhibitors may represent a new strategy for treating pheochromocytomas.

The psychostimulant properties of methamphetamine (METH) are associated with an increase in extracellular dopamine (DA) levels in the brain, via facilitation of DA's release from pre-synaptic nerve terminals and inhibition of its reuptake through DA transporter. Recently, we have demonstrated that tumor necrosis factor-alpha (TNF-alpha) increases DA uptake and inhibits METH dependence. Moreover, we have clarified 'shati' identified in the nucleus accumbens of mice treated with METH is involved in METH dependence. In the present study, we investigated the effects of TNF-alpha on DA uptake in PC12 cells and established a PC12 cell line transfected with a vector containing shati cDNA to examine the precise mechanism behind the role of shati in DA uptake. Moreover, we examined the relationship between shati and TNF-alpha. TNF-alpha increased DA uptake via the mitogen-activated protein kinase kinase pathway and inhibited the METH-induced decrease in DA uptake in PC12 cells. Transfection of the vector containing shati cDNA into PC12 cells, induced the expression of shati and TNF-alpha mRNA, accelerated DA uptake, and inhibited the METH-induced decrease in DA uptake. These results suggest that the functional roles of shati in METH-regulated behavioral changes are mediated through inhibition of the METH-induced decrease in DA uptake via TNF-alpha.

The replication of herpes simplex virus (HSV) in epithelial cells, and during reactivation from latency in sensory neurons, depends on a ubiquitous cellular protein called host cell factor (HCF). The HSV transactivator, VP16, which initiates the viral replicative cycle, binds HCF as do some other cellular proteins. Of these, the neuronal transcription factor Zhangfei suppresses the ability of VP16 to initiate the replicative cycle. It also suppresses Luman, another cellular transcription factor that binds HCF. Interactions of nerve growth factor (NGF) and its receptor tropomyosin-related kinase (trkA) appear to be critical for maintaining HSV latency. Because the neuronal transcription factor Brn3a, which regulates trkA expression, has a motif for binding HCF, we investigated if Zhangfei had an effect on its activity. We found that Brn3a required HCF for activating the trkA promoter and Zhangfei suppressed its activity in non-neuronal cells. However, in neuron-like NGF-differentiated PC12 cells, both Brn3a and Zhangfei activated the trkA promoter and induced the expression of endogenous trkA. In addition, capsaicin, a stressor, which activates HSV in in vitro models of latency, decreased levels of Zhangfei and trkA transcripts in NGF-differentiated PC12 cells.

We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 microM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.

Effects of diazoxon on the gene and protein expression of nicotinic acetylcholine receptors (nAChR) were evaluated in PC12 cells. Cells were exposed to 100 microM diazoxon for 48 h in the presence versus absence of nAChR agonists or antagonists. Diazoxon significantly inhibited AChE activity in the cells. At the mRNA level, transcripts of the alpha4 and beta2 subunits of nAChR were significantly reduced in cells exposed to diazoxon, but there was no change in alpha7 subunit mRNA content. Diazoxon exposure also significantly reduced the protein levels of both alpha4 and beta2 nAChR subunits. Treatment with nicotine (10 microM) or with the nicotinic receptor antagonists, mecamylamine (10 microM) or dihydro-beta-erythroidine (DHbetaE) (5 microM) efficiently prevented the diazoxon-induced reduction in alpha4 and beta2 nAChR mRNA and protein in PC12 cells, but carbamaylcholine, a weak nAChR agonist, was ineffective. These data suggest that alpha4beta2 nAChRs are involved in diazoxon-related toxicity and that nicotinic receptor antagonists could play a protective role against organophosphate-related damage.

The effects of gamma-hexachlorocyclohexane, one of the endocrine disrupters, widely used for agricultural and medicinal purpose, on apoptosis in PC12 cells were investigated using western blotting analysis and reverse transcriptase-DNA polymerase chain reaction (RT-PCR) method. Apoptosis is a fundamental process necessary for development of individuals and organs. Although gamma -HCH at high concentration did not have an effect on cell viability and apoptosis, DNA fragmentation was slightly enhanced, and apoptotic factors; Bax, Bad, cytochrome c and caspase-3 showed tendency to increase by the addition of a low dose of gamma-HCH to the cell medium. However these changes were not statistically significant. It was concluded that gamma -HCH did not affect on apoptosis in the PC12 cell line system, although gamma -HCH has been reported to induce apoptosis in other cell lines.

Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling. In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT(1)) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT(1A) receptor (AT(1)R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA(A) receptor, as a binding partner for the AT(1)R. Interaction of GABARAP with the AT(1)R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT(1)R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT(1)R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT(1)R in PC-12 cells. Cotransfection of GABARAP with an AT(1)R fluorescent fusion protein increased PC-12 cell surface expression of the AT(1)R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT(1)R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT(1)R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT(1)R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT(1)R to the plasma membrane.

Background: Oxidative metabolism, resulting in the formation of hydroxylated polybrominated diphenyl ether (PBDE) metabolites, may enhance the neurotoxic potential of brominated flame retardants., Objective: Our objective was to investigate the effects of a hydroxylated metabolite of 2,2',4,4'-tetra-bromodiphenyl ether (BDE-47; 6-OH-BDE-47) on changes in the intracellular Ca2+ concentration ([Ca2+]i) and vesicular catecholamine release in PC12 cells., Methods: We measured vesicular catecholamine release and [Ca2+]i using amperometry and imaging of the fluorescent Ca2+-sensitive dye Fura-2, respectively., Results: Acute exposure of PC12 cells to 6-OH-BDE-47 (5 microM) induced vesicular catecholamine release. Catecholamine release coincided with a transient increase in [Ca2+]i, which was observed shortly after the onset of exposure to 6-OH-BDE-47 (120 microM). An additional late increase in [Ca2+]i was often observed at > or =1 microM 6-OH-BDE-47. The initial transient increase was absent in cells exposed to the parent compound BDE-47, whereas the late increase was observed only at 20 microM. Using the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and thapsigargin to empty intracellular Ca2+ stores, we found that the initial increase originates from emptying of the endoplasmic reticulum and consequent influx of extracellular Ca2+, whereas the late increase originates primarily from mitochondria., Conclusion: The hydroxylated metabolite 6-OH-BDE-47 is more potent in disturbing Ca2+ homeostasis and neurotransmitter release than the parent compound BDE-47. The present findings indicate that bioactivation by oxidative metabolism adds considerably to the neurotoxic potential of PBDEs. Additionally, based on the observed mechanism of action, a cumulative neurotoxic effect of PBDEs and ortho-substituted polychlorinated biphenyls on [Ca2+]i cannot be ruled out.

The genetic background and the pathogenesis of familial Parkinson's disease (PD) have not been fully elucidated. Two missense mutations in the alpha-synuclein gene, A30P and A53T, have been linked to familial PD. Increasing evidence suggests the involvement of alpha-synuclein, the dopamine transporter (DAT), and neurotoxins in the pathogenesis of PD, but their relationships to the death of nigral cells remains poorly understood. In the present study, we used the PC12 cell line, a well recognized model of the nigral cell, to investigate the effects of overexpression of wild-type (WT) and mutant human alpha-synuclein on MPP+-induced neurotoxicity. We found that overexpression of mutant alpha-synuclein enhanced the toxicity of MPP+ to PC12 cells and elevated intracellular reactive oxygen species (ROS) levels, whereas overexpression of WT alpha-synuclein protected PC12 cells against MPP+ toxicity and lowered ROS levels. Furthermore, assays of 131I-FP-beta-CIT binding with DAT membrane fractions showed that WT and mutant alpha-synuclein had different effects on the expression of DAT on the cell membrane following exposure to MPP+. WT alpha-synuclein reduced the toxic effect of MPP+ by facilitating DAT internalization, while both A30P and A53T alpha-synuclein aggravated the toxic effect of MPP+ by reducing DAT internalization. These data indicate that alpha-synuclein regulates ROS levels and DAT surface expression in dopaminergic neurons, and thus changes the response of these cells to MPP+.

Many studies of Ca2+ signaling use PC12 cells, yet the balance of Ca2+ clearance mechanisms in these cells is unknown. We used pharmacological inhibition of Ca2+ transporters to characterize Ca2+ clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), plasma membrane Ca2+ ATPase (PMCA), and Na+/Ca2+ exchanger (NCX) account for almost all Ca2+ clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of Ca2+ clearance possessing pharmacological characteristics consistent with secretory pathway Ca2+ ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall Ca2+ transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released Ca2+ instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed Ca2+ clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous Ca2+ binding ratio of PC12 cell cytoplasm, which we measured to be 268 +/- 85. Our results indicate that Ca2+ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different Ca2+ transport mechanisms.

We studied the effects of antiparkinsonian drug himantane (acute and subchronic administration) on the content of dopamine transporter protein DAT in rat striatum ex vivo and on the content of DTA in cultured PC-12 cells (10(-5) - 10(-7) M, the preparation was added to the incubation medium once or 7 times). The preparation significantly reduced the content of DAT protein both ex vivo and in vitro.

We found that ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] caused phosphorylation of mitogen-activated protein kinase (MAPK), followed by expression of neurofilament-M, a neuron-specific protein, in cultured PC12 rat pheochromocytoma cells. The ebselen-induced MAPK activation was suppressed by U0126, an inhibitor of MAPK kinase (MEK1/2), but not by K252a, a selective inhibitor of Trk family tyrosine kinases; AG1478, an antagonist of epidermal growth factor receptor (EGFR); pertussis toxin, an inhibitor of Gi/o; or GP antagonist-2A, an inhibitor of Gq. Furthermore, we observed that N-acetyl-L-cysteine, an inhibitor of tyrosine kinases, suppressed ebselen-induced MAPK activation and buthionine sulfoximine, an activator of protein tyrosine phosphatases, enhanced the effect, indicating that ebselen activated MEK1/2 through one or more tyrosine kinases. Based on these results, we propose that ebselen stimulated intracellular tyrosine kinase activity, thus activating a MAPK cascade (tyrosine kinase-MEK1/2-ERK1/2) in PC12 cells and that this activation resulted in their neuronal differentiation., ((c) 2007 Wiley-Liss, Inc.)

Depolarization, growth factors, neurotrophins, and other stimuli induce expression of immediate early genes (IEGs) in neurons. We identified a subset of IEGs, IPD-IEGs, which are induced preferentially by depolarization, but not by neurotrophins or growth factors, in PC12 cells. The "promiscuous" IEGs Egr1 and c-fos, induced by growth factors and neurotrophins, in addition to depolarization, require activation of the MAP kinase signaling pathway for induction in response to KCl depolarization in PC12 cells; MEK1/2 inhibitors block KCl-induced Egr1 and c-fos expression. In contrast, MEK1/2 inhibition has no effect on KCl-induced expression of the known IPD-IEGs in PC12 cells. Additional "candidate" IDP-IEGs were identified by a microarray comparison of genes induced by KCl in the presence vs. the absence of an MEK1/2 inhibitor in PC12 cells. Northern blot analyses demonstrated that representative newly identified candidate IPD-IEGs, as with the known IPD-IEGs, are also induced by a MAP kinase- independent pathway in response to depolarization, both in PC12 cells and in rat primary cortical neurons. Nerve growth factor and epidermal growth factor are unable to induce the expression of the Crem/Icer, Nur77, Nor1, Rgs2, Dusp1 (Mkp1), and Dscr1 genes in PC12 cells, validating their identification as IPD-IEGs. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII), calcineurin, or protein kinase A (PKA) activity prevents KCl-induced IPD-IEG mRNA accumulation, suggesting that the IPD-IEG genes are induced by depolarization in neurons via a combination of calcineurin/PKA- and CaMKII-dependent pathways., ((c) 2007 Wiley-Liss, Inc.)

Me-Hg and PCB153 are known neurotoxic contaminants which tend to accumulate in food, particularly in fish. Aim of this study was to perform asynchronous and combined exposure to Me-Hg and PCB153 in a neuronal rat cell line (PC12) to better characterise the antagonism observed at some combination concentrations. PC12 cells were treated with three concentrations of Me-Hg (0.1-0.5-1.0 microM) and PCB153 at one concentration (175 microM) in single and combined asynchronous exposures, using viability (MTT assay) as end-point. At all concentrations used, a statistically significant antagonistic effect was observed when Me-Hg preceded PCB153 exposure, while effect was additive when PCB153 preceded Me-Hg exposure. The antagonism is particularly evident at low concentrations of Me-Hg (0.1 microM). In conclusion, combined asynchronous exposure showed that whereas Me-Hg can modulate PCB153 toxicity, the opposite seems not to be true. Therefore, the use of asynchronous exposure could be a promising approach to study the mechanisms of toxicity of binary mixtures.

We hypothesized that the neuroprotective role of the standardized Ginkgo biloba (Ginkgoaceae) extract EGb 761 under hypoxic conditions might be associated with its function to increase HIF-1 activity based on the fact that oxygen availability is crucial for cellular metabolism and viability and that HIF-1 plays an essential role in cellular oxygen homeostasis under hypoxic conditions. In this study, we therefore investigated the effects of ginkgolides, the main constituent of the non-flavone fraction of EGb 761, on the content and activity of HIF-1alpha, a key factor to determine HIF-1 activity, in hypoxic PC12 cells induced by cobalt chloride. Our data demonstrated that ginkgolides have a significant protective role against hypoxia-induced injury in the PC12 cells. The findings also strongly support our hypothesis that the protective role of ginkgolides is due to the up-regulation of HIF-1alpha protein expression and modification through the ginkgolides-induced activation of the p42/p44 MAPK pathway. In addition, it was evident that ginkgolides could significantly increase the HIF-1 DNA binding activity, which might also be associated with the protective effects of ginkgolides by promoting the expression of target genes of HIF-1 under hypoxic conditions., ((c) 2007 Wiley-Liss, Inc.)

The receptor for advanced glycation end products (RAGE) binds amyloid peptides with high affinity. Soluble RAGE-like peptides and Abeta-like peptides occur in relatively high concentrations in the circulation of individuals with Alzheimer's disease. Protein complexes with epitopes for both Abeta and RAGE are also present. At physiological concentrations, forms of Abeta have different, but relatively low potencies as cytotoxicants in neural cells in culture. The purpose of this study was to determine whether a synthetic peptide complex composed of Abeta(1-42) and RAGE(23-54), a conserved N-terminal fragment of RAGE, exhibited increased cytotoxicity in comparison with the constituent peptides. Western analysis indicated that Abeta(1-42) and RAGE(23-54) remained primarily in their original low molecular weight states (4-6 kDa) during the maintenance of the individual peptides (37 degrees C) in water from 1 to 4 weeks. In contrast, over the same maintenance periods the combined Abeta(1-42) and RAGE(23-54) peptides shifted to higher molecular weight complexes (up to 80-120 kDa). Protein complexes of similar molecular weights with epitopes for Abeta and RAGE antibodies were identified in human plasma. Incubation of differentiated PC-12 cells with 10-100 microM Abeta(1-42) or with RAGE(23-54) resulted in concentration-dependent decreases in cell viability. The cytotoxicity of each peptide was slightly enhanced by the progressive maintenance of Abeta(1-42) and RAGE(23-54) in water over 3 weeks prior to the assay. Under the same conditions, the Abeta(1-42) - RAGE(23-54) complex became significantly more cytotoxic. These results suggest that the formation of soluble Abeta-RAGE complexes in Alzheimer's disease could represent a mechanism for enhancing the neurotoxicity of amyloid peptides.

Ratiometric fluorescent indicators are becoming increasingly prevalent in many areas of biology. They are used for making quantitative measurements of intracellular free calcium both in vitro and in vivo, as well as measuring membrane potentials, pH, and other important physiological variables of interest to researchers in many subfields. Often, functional changes in the fluorescent yield of ratiometric indicators are small, and the signal-to-noise ratio (SNR) is of order unity or less. In particular, variability in the denominator of the ratio can lead to very poor ratio estimates. We present a statistical optimization method for objectively detecting and estimating ratiometric signals in dual-wavelength measurements of fluorescent, ratiometric indicators that improves on standard methods. With the use of an appropriate statistical model for ratiometric signals and by taking the pixel-pixel covariance of an imaging dataset into account, we are able to extract user-independent spatiotemporal information that retains high resolution in both space and time.

The catecholamine-secreting PC12 cell line derived from the rat adrenal medulla has long been considered a model system for neurosecretion and neuronal differentiation. PC12 cells contain a large number of secretory granules (otherwise known as large dense-core vesicles) for storage of small molecules, processing enzymes, neuropeptides, and peptide hormones. Secretory granule exocytosis in PC12 cells is tightly regulated by calcium and occurs in response to a secretagogue. This unit provides protocols for maintenance and transfection of PC12 cells. Several secretion assays are described to measure the release of secretory granule cargo molecules by detection of radioactive catecholamine, or by immunochemical or chemiluminescence detection of transfected regulated secretory proteins., ((c) 2007 by John Wiley & Sons, Inc.)