Rat Pheochromocytoma PC12 Cells Research Articles

Ganglioside GQ1b induces dopamine release through the activation of Pyk2.

Growing evidence indicates that GQ1b, one of the gangliosides members, contributes to synaptic transmission and synapse formation. Previous studies have shown that GQ1b could enhance depolarization induced neurotransmitter release, while the role of GQ1b in asynchronous release is still largely unknown. Here in our result, we found low concentration of GQ1b, but not GT1b or GD1b (which were generated from GQ1b by plasma membrane-associated sialidases), evoked asynchronous dopamine (DA) release from both clonal rat pheochromocytoma PC12 cells and rat striatal slices significantly. The release peaked at 2 min after GQ1b exposure, and lasted for more than 6 min. This effect was caused by the enhancement of intracellular Ca(2+) and the activation of Pyk2. Inhibition of Pyk2 by PF-431396 (a dual inhibitor of Pyk2 and FAK) or Pyk2 siRNA abolished DA release induced by GQ1b. Moreover, Pyk2 Y402, but not other tyrosine site, was phosphorylated at the peaking time. The mutant of Pyk2 Y402 (Pyk2-Y402F) was built to confirm the essential role of Y402 activation. Further studies revealed that activated Pyk2 stimulated ERK1/2 and p-38, while only the ERK1/2 activation was indispensable for GQ1b induced DA release, which interacted with Synapsin I directly and led to its phosphorylation, then depolymerization of F-actin, thus contributed to DA release. In conclusion, low concentration of GQ1b is able to enhance asynchronous DA release through Pyk2/ERK/Synapsin I/actin pathway. Our findings provide new insights into the role of GQ1b in neuronal communication, and implicate the potential application of GQ1b in neurological disorders.

Structure-Activity Relationship Study of the Neuritogenic Potential of the Glycan of Starfish Ganglioside LLG-3 (‡).

LLG-3 is a ganglioside isolated from the starfish Linchia laevigata. To clarify the structure-activity relationship of the glycan of LLG-3 toward rat pheochromocytoma PC12 cells in the presence of nerve growth factor, a series of mono- to tetrasaccharide glycan derivatives were chemically synthesized and evaluated in vitro. The methyl group at C8 of the terminal sialic acid residue was crucial for neuritogenic activity, and the terminal trisaccharide moiety was the minimum active motif. Furthermore, the trisaccharide also stimulated neuritogenesis in human neuroblastoma SH-SY5Y cells via mitogen-activated protein kinase (MAPK) signaling. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rapidly induced by adding 1 or 10 nM of the trisaccharide. The ratio of phosphorylated ERK to ERK reached a maximum 5 min after stimulation, and then decreased gradually. However, the trisaccharide did not induce significant Akt phosphorylation. These effects were abolished by pretreatment with the MAPK inhibitor U0126, which inhibits enzymes MEK1 and MEK2. In addition, U0126 inhibited the phosphorylation of ERK 1/2 in response to the trisaccharide dose-dependently. Therefore, we concluded that the trisaccharide promotes neurite extension in SH-SY5Y cells via MAPK/ERK signaling, not Akt signaling.

Partial Protection of PC12 Cells from Cellular Stress by Low-Dose Sodium Nitroprusside Pre-treatment.

The PC12 rat pheochromocytoma cell line is an in vitro model system widely used for the investigation of intracellular signaling events contributing to neuronal differentiation and cell death. We found earlier that the nitric oxide donor compound sodium nitroprusside (SNP) induced apoptosis of PC12 cells if it was applied in high concentration (400µM). Yoshioka et al. (J Pharmacol Sci 101:126-134, 2006) reported that cell death evoked by cytotoxic concentrations of SNP could be prevented by a 100µM SNP pre-treatment in a murine macrophage cell line. The apoptosis caused by toxic-dose SNP treatment (400µM) could be partially overcome in PC12 cells as well by the low-dose SNP pre-treatment. The partial inhibition of apoptosis was accompanied by reduced phosphorylation of certain proteins (such as stress-activated protein kinases, the p53, and the eIF2α proteins), decreased caspase activation, and less intense internucleosomal DNA fragmentation. The 100µM SNP pre-treatment reduced the pro-apoptotic potential of certain other stress stimuli (serum withdrawal, cisplatin and tunicamycin treatments) as well, although the underlying biochemical changes were not entirely uniform. On the contrary, the 100µM SNP pre-treatment was unable to prevent cell death caused by the protein synthesis inhibitor anisomycin. Further clarification of the above-mentioned processes may be important in understanding the mechanisms by which mild nitrosative stress protects cells against certain forms of cellular stress conditions.

Tanshinone II A, a multiple target neuroprotectant, promotes caveolae-dependent neuronal differentiation

Neuron loss is one fundamental features of neurodegenerative diseases. Stimulating endogenous neurogenesis, especially neuronal differentiation, might potentially provide therapeutic effects to these diseases. In this study, tanshinone II A (TIIA), a multiple target neuroprotectant, was demonstrated to promote dose-dependent neuronal differentiation in three cell models of immortalized C17.2 neuronal stem cells, rat embryonic cortical neural stem cells (NSCs) and rat PC12 pheochromocytoma cells. In particular, TIIA exerted promising effects on NSCs even at the dose of 3nM. In PC12 cells, TIIA activated mitogen-activated protein kinase 42/44 (MAPK42/44) and its downstream transcription factor, cAMP response element-binding protein (CREB). In addition, TIIA up-regulated the expressions of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). The MEK inhibitor and the antagonist to the receptors of NGF and BDNF could partially attenuate the differentiation effects, indicating that MAPK42/44 mediated BDNF and NGF signals were involved in TIIA's differentiation effects. Caveolin-1 (CAV-1), the major functional protein of membrane caveolae, plays critical roles in the endocytosis of exogenous materials. CAV1, which was activated by TIIA, might help TIIA transport across cell membrane to initiate its differentiation effects. It was proven by the evidences that suppressing the function of caveolin inhibited the differentiation effects of TIIA. Therefore, we concluded that TIIA promoted neuronal differentiation partially through MAPK42/44 mediated BDNF and NGF signals in a caveolae-dependent manner.

Essential Oils from the Medicinal Herbs Upregulate Dopamine Transporter in Rat Pheochromocytoma Cells.

The dopamine transporter (DAT) protein, a component of the dopamine system, undergoes adaptive neurobiological changes from drug abuse. Prevention of relapse and reduction of withdrawal symptoms are still the major limitations in the current pharmacological treatments of drug addiction. The present study aimed to investigate the effects of essential oils extracted from Elsholtzia ciliata, Shinchim, Angelicae gigantis Radix, and Eugenia caryophyllata, well-known traditional Korean medicines for addiction, on the modulation of dopamine system in amphetamine-treated cells and to explore the possible mechanism underlying its therapeutic effect. The potential cytotoxic effect of essential oils was evaluated in PC12 rat pheochromocytoma cells using cell viability assays. Quantification of DAT, p-CREB, p-MAPK, and p-Akt was done by immunoblotting. DAT was significantly reduced in cells treated with 50 μM of amphetamine in a time-dependent manner. No significant toxicity of essential oils from Elsholtzia ciliata and Shinchim was observed at doses of 10, 25, and 50 μg/mL. However, essential oils from A. gigantis Radix at a dose of 100 μg/mL and E. caryophyllata at doses of 50 and 100 μg/mL showed cytotoxicity. Treatment with GBR 12909, a highly selective DAT inhibitor, significantly increased DAT expression compared with that of amphetamine only by enhancing phosphorylation of mitogen-activated protein kinases (MAPK) and Akt. In addition, essential oils effectively induced hyperphosphorylation of cyclic-AMP response element-binding protein (CREB), MAPK, and Akt, which resulted in DAT upregulation. Our study implies that the essential oils may rehabilitate brain dopamine function through increased DAT availability in abstinent former drug users.

Effect of mesoporous silica nanoparticles on cell viability and markers of oxidative stress

In the recent years, the use of mesoporous silica nanoparticles (MSNs) has been extended in biomedical fields such as cancer therapy, drug and gene delivery, biosensors, and enzyme immobilization. Although nanomaterials are currently being widely used in modern technology, there is a lack of information regarding to the health and environmental implications of manufactured nanomaterials. In the present study, the effects of MSNs and surface functionalized MSNs on cell viability, markers of oxidative damages (mainly intracellular reactive oxygen species (ROS) formation), and oxidative DNA damage were investigated in vitro in rat pheochromocytoma PC12 cells. Following exposure of these nanoparticles (1.95–1000 µg/mL) to PC12 cells for 12 and 24 h, no significant reduction of cell viability was observed compared with control. Moreover, ROS formation and oxidative DNA damage were not significantly changed by these nanoparticles even at high concentrations or prolong exposures. In conclusion, the results showed that neither MSNs nor functionalized MSNs exhibited any remarkable in vitro toxic properties in PC12 cells even at high concentration.

Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca2+ channels

Autism is a neurodevelopmental psychiatric disorder characterized by impaired reciprocal social interaction, disrupted communication, and restricted and stereotyped patterns of interests. Autism is known to have a strong genetic component. Although mutations in several genes account for only a small proportion of individuals with autism, they provide insight into potential biological mechanisms that underlie autism, such as dysfunction in Ca2+ signaling, synaptic dysfunction, and abnormal brain connectivity. In autism patients, two mutations have been reported in the Rab3 interacting molecule 3 (RIM3) gene. We have previously demonstrated that RIM3 physically and functionally interacts with voltage-dependent Ca2+ channels (VDCCs) expressed in neurons via the β subunits, and increases neurotransmitter release. Here, by introducing corresponding autism-associated mutations that replace glutamic acid residue 176 with alanine (E176A) and methionine residue 259 with valine (M259V) into the C2B domain of mouse RIM3, we demonstrate that both mutations partly cancel the suppressive RIM3 effect on voltage-dependent inactivation of Ba2+ currents through P/Q-type CaV2.1 recombinantly expressed in HEK293 cells. In recombinant N-type CaV2.2 VDCCs, the attenuation of the suppressive RIM3 effect on voltage-dependent inactivation is conserved for M259V but not E176A. Slowing of activation speed of P/Q-type CaV2.1 currents by RIM3 is abolished in E176A, while the physical interaction between RIM3 and β subunits is significantly attenuated in M259V. Moreover, increases by RIM3 in depolarization-induced Ca2+ influx and acetylcholine release are significantly attenuated by E176A in rat pheochromocytoma PC12 cells. Thus, our data raise the interesting possibility that autism phenotypes are elicited by synaptic dysfunction via altered regulation of presynaptic VDCC function and neurotransmitter release.

Regulation of NGF‐driven neurite outgrowth by Ins(1,4,5)P3 kinase is specifically associated with the two isoenzymes Itpka and Itpkb in a model of PC12 cells

Four inositol phosphate kinases catalyze phosphorylation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 ] to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4 ]: these enzymes comprise three isoenzymes of inositol 1,4,5-trisphosphate 3-kinase (Itpk), referred to as Itpka, Itpkb and Itpkc, and the inositol polyphosphate multikinase (IPMK). The four enzymes that act on Ins(1,4,5)P3 are all expressed in rat pheochromocytoma PC12 cells, a model that is used to study neurite outgrowth induced by nerve growth factor (NGF). We compared the effect of over-expression of the four GFP-tagged kinases on NGF-induced neurite outgrowth. Our data show that over-expression of the Itpka and Itpkb isoforms inhibits NGF-induced neurite outgrowth, but over-expression of Itpkc and IPMK does not. Surprisingly, over-expression of the N-terminal F-actin binding domain of Itpka, which lacks catalytic activity, was as effective at inhibiting neurite outgrowth as the full-length enzyme. Neurite length was also significantly decreased in cells over-expressing Itpka and Itpkb but not Itpkc or IPMK. This result did not depend on the over-expression level of any of the kinases. PC12 cells over-expressing GFP-tagged kinase-dead mutants Itpka/b have shorter neurites than GFP control cells. The decrease in neurite length was never as pronounced as observed with wild-type GFP-tagged Itpka/b. Finally, the percentage of neurite-bearing cells was increased in cells over-expressing the membranous type I Ins(1,4,5)P3 5-phosphatase. We conclude that Itpka and Itpkb inhibit neurite outgrowth through both F-actin binding and localized Ins(1,4,5)P3 3-kinase activity. Itpkc and IPMK do not influence neurite outgrowth or neurite length in this model.

Induction of Neurite Outgrowth in PC12 Cells Treated with Temperature-Controlled Repeated Thermal Stimulation

To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine.

Low Doses of Camptothecin Induced Hormetic and Neuroprotective Effects in PC12 Cells.

Hormetic response is an adaptive mechanism for a cell or organism surviving in an unfavorable environment. It has been an intriguing subject of researches covering a broad range of biological and medical disciplines, in which the underlying significance and molecular mechanisms are under intensive investigation. In the present study, we demonstrated that topoisomerase I inhibitor camptothecin (CPT), a potent anticancer agent, induced an obvious hormetic response in rat pheochromocytoma PC12 cells. Camptothecin inhibited PC12 cell growth at relative high doses as generally acknowledged while stimulated the cell growth by as much as 39% at low doses. Moreover, low doses of CPT protected the cells from hydrogen peroxide (H2O2)-induced cell death. Phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathways were reported playing pivotal roles in protecting cells from oxidative stress. We observed that these 2 pathways were upregulated by low doses of CPT, as evidenced by increased levels of phosphorylated PI3K, phosphorylated Akt, phosphorylated mammalian target of rapamycin, Nrf2, and HO-1; and abolishment of the growth-promoting and neuroprotective effects of CPT by LY294002, a PI3K inhibitor. These results suggest that the hormetic and neuroprotective effects of CPT at low doses on PC12 cells were attributable, at least partially, to upregulated PI3K/Akt and Nrf2/HO-1 pathways.

Luminescence Lifetime Imaging Microscopy For Analysis Of Oxygen In The Cancer Cell

Molecular oxygen (O2), the key component of aerobic metabolism, plays important roles in normal and cancer cells and tissues. A real‐time and quantitative O2 imaging in 3D cell models would allow to discriminate complex effects of hypoxia on cell function in complex (heterogeneous) cell populations at subcellular resolution. Here, we used luminescence lifetime imaging (FLIM, PLIM) to develop and evaluate phosphorescent O2 probes with 3D spheroid and multi‐cellular aggregate models. We tested rat embryonic neurosphere culture with cationic nanoparticle O2 probes (MM2, NanO2) and developed O2 imaging protocol, multiplexed with analysis of distribution of specific protein markers such as HIF‐2α. To address the problem of efficient in‐depth staining, we designed two novel intracellular O2 probes: small molecule Pt‐Glc and nanoparticle PA2. Both probes displayed high analytical performance (brightness, photostability, O2 calibration) and were evaluated with rat pheochromocytoma PC12 and human colon cancer HCT116 cell aggregates. Resting cells cultured in 3D aggregates of >50 μm size displayed significant deoxygenation even under ambient atmospheric O2 levels. Still, they showed high viability and experienced different levels of oxygenation, upon treatment with mitochondrial uncouplers, inhibitors or pharmacological drugs. This data confirms the importance of probing of cellular O2 for experiments with 3D cell models. When combined with other live cell imaging dyes, such as markers of necrosis and apoptosis, the method allows for multi‐parameter quantitative assessment of cell physiology within relevant 3D micro‐environment.Supported by Science Foundation Ireland (SFI) grant 13/SIRG/2144.

HIF-1α activation by intermittent hypoxia requires NADPH oxidase stimulation by xanthine oxidase.

Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O2 saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O2 for 30 sec) and normoxia (21% O2 for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47phox and p67phox to the plasma membrane and their interaction with gp91phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.

Icariin Prevents Amyloid Beta-Induced Apoptosis via the PI3K/Akt Pathway in PC-12 Cells

Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum that exerts a variety of pharmacological activities and shows promise in the treatment and prevention of Alzheimer's disease. In this study, we investigated the neuroprotective effects of icariin against amyloid beta protein fragment 25–35 (Aβ 25–35) induced neurotoxicity in cultured rat pheochromocytoma PC12 cells and explored potential underlying mechanisms. Our results showed that icariin dose-dependently increased cell viability and decreased Aβ 25–35-induced apoptosis, as assessed by MTT assay and Annexin V/propidium iodide staining, respectively. Results of western blot analysis revealed that the selective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 suppressed icariin-induced Akt phosphorylation, suggesting that the protective effects of icariin are associated with activation of the PI3K/Akt signaling pathway. LY294002 also blocked the icariin-induced downregulation of proapoptotic factors Bax and caspase-3 and upregulation of antiapoptotic factor Bcl-2 in Aβ 25–35-treated PC12 cells. These findings provide further evidence for the clinical efficacy of icariin in the treatment of Alzheimer's disease.

Nucleofection of rat pheochromocytoma PC-12 cells with human mutated beta-amyloid precursor protein gene (APP-sw) leads to reduced viability, autophagy-like process, and increased expression and secretion of beta amyloid.

Pheochromocytoma PC-12 cells are immune to physiological stimuli directed to evoke programmed cell death. Besides, metabolic inhibitors are incapable of sensitizing PC-12 cells to extrinsic or intrinsic apoptosis unless they are used in toxic concentrations. Surprisingly, these cells become receptive to cell deletion after human APP-sw gene expression. We observed reduced cell viability in GFP vector + APP-sw-nucleofected cells (drop by 36%) but not in GFP vector − or GFP vector + APP-wt-nucleofected cells. Lower viability was accompanied by higher expression of Aβ 1-16 and elevated secretion of Aβ 1-40 (in average 53.58 pg/mL). At the ultrastructural level autophagy-like process was demonstrated to occur in APP-sw-nucleofected cells with numerous autophagosomes and multivesicular bodies but without autolysosomes. Human APP-sw gene is harmful to PC-12 cells and cells are additionally driven to incomplete autophagy-like process. When stimulated by TRAIL or nystatin, CLU protein expression accompanies early phase of autophagy.

The pseudophosphatase MK-STYX induces neurite-like outgrowths in PC12 cells.

The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and implicate this pseudophosphatase as a regulator of neuronal differentiation.

Neurotropin promotes NGF signaling through interaction of GM1 ganglioside with Trk neurotrophin receptor in PC12 cells

Activation of the high-affinity nerve growth factor (NGF) receptor Trk occurs through multiple processes consisted of translocation and clustering within the plasma membrane lipid rafts, dimerization and autophosphorylation. Here we found that a nonprotein extract of inflamed rabbit skin inoculated with vaccinia virus (Neurotropin®) enhanced efficiency of NGF signaling. In rat pheochromocytoma PC12 cells overexpressing Trk (PCtrk cells), Neurotropin augmented insufficient neurite outgrowth observed at suboptimal concentration of NGF (2ng/mL) in a manner depending on Trk kinase activity. Cellular exposure to Neurotropin resulted in an accumulation of Trk-GM1 complexes without affecting dimerization or phosphorylation states of Trk. Following NGF stimulation, Neurotropin significantly facilitated the time course of NGF-induced Trk autophosphorylation. These observations provide a unique mechanism controlling efficiency of NGF signaling, and raise the therapeutic potential of Neurotropin for various neurological conditions associated with neurotrophin dysfunction.

In-situ detection of neurotransmitter release from PC12 cells using Surface Enhanced Raman Spectroscopy

Rat pheochromocytoma PC12 cells have frequently been used as a dopaminergic neuron model due to their various functions, including the synthesis, storage, and secretion of catecholamines. Furthermore, PC12 cells release a measurable amount of dopamine (DA) in response to some chemicals. PC12 cells are thus considered to be one of the most common invitro models for studying neurotransmitter release. Here, we applied Surface-enhanced Raman Spectroscopy (SERS) to determine with high sensitivity the in-situ short-time effects of cisplatin (cisdiamine- dichloroplatinum), bisphenol-A, and cyclophosphamide on the extracellular DA level released from PC12 cells. In addition, using the SERS technique, changes in the biochemical composition of the PC12 cell lysates were investigated to determine the intracellular DA level. Gold nano-patterned substrates were fabricated based on electrochemical deposition of Au nanorods onto ITO substrates; these substrates were then used as SERS-active surfaces. The Raman spectroscopy results demonstrated that the changes in the Raman spectra depending on the treatment agent were in agreement with the HPLC results on the extracellular DA level. Therefore, the SERS technique can overcome the limitations of other detection techniques, and can be used with cellular nanoarrays to study the effect of a wide range of chemicals.

Echinacoside inhibits amyloid fibrillization of HEWL and protects against Aβ-induced neurotoxicity

Amyloid protein depositions play crucial roles in a variety of degenerative disorders composing amyloidosis. There is a great interest in developing small molecule inhibitors of amyloidogenic processes. We examined the inhibitory effects of echinacoside (ECH) with different concentrations and at different fiber-forming stages in vitro utilizing the hen egg-white lysozyme (HEWL) model system. We also evaluated the antioxidant capacity of ECH by using elimination tests for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl (HO) free radicals. We investigated the protection provided by ECH against neurotoxicity induced by β-amyloid protein (Aβ). Through spectroscopic analyses, electron microscopy, cell viability assay, and hemolysis assay, we found that ECH dose dependently inhibited HEWL aggregation, and this inhibition occurred in different fiber-forming stages. ECH could also scavenge the DPPH and OH free radicals in a concentration-dependent manner. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2′,7′-dichlorodihydrofluoresceindiacetate (DCFH-DA) fluorescent measurement results indicated that ECH could increase viability of rat pheochromocytoma PC12 cells injured by Aβ and suppress the increase in intracellular reactive oxygen species (ROS) triggered by Aβ. The present study findings facilitate a better understanding of the interaction between ECH and amyloid-forming proteins and also shed light on the protection of ECH against amyloid fibril-induced neuronal cell death.

Structure-based study of antiamyloidogenic cyclic d,l-α-peptides

Protein misfolding and aggregation are the hallmarks of many devastating diseases. We have previously shown that cyclic d,l-α-peptide CP-2 reacts and stabilizes less toxic forms of amyloid β (Aβ), and protects the cells from Aβ-induced toxicity. Here, we performed extensive structure-based studies on CP-2 to elucidate the contribution of each residue to the total antiamyloidogenic activity and determine the interactions that are involved between CP-2 and Aβ. We showed that the hydrophobicity of CP-2 analogs correlates with their antiamyloidogenic potency, however, aromatic interactions are even more important for this activity. The antiamyloidogenic activity of CP-2 analogs also correlates with their ability to self-assemble, as shown by the critical micelle concentration measurements. The cell survival studies performed on rat pheochromocytoma PC-12 cells suggest that incorporation of an additional aromatic residue to the CP-2's sequence increases its protective effect against Aβ42-induced toxicity.

Nerve cell differentiation using constant and programmed electrical stimulation through conductive non-functional graphene nanosheets film

Electrical signal is one of the most important elctrophysiological phenomena and is widely used in electrogensis and electrodiagnosis. The use of appropriate external electrical stimulation (ES) to in situ modify cellular behavior can desirably promote tissue regeneration. Considering its high electron mobility and surface area, graphene could become a promising conductive scaffold in biomedical applications. In this study, a programmed ES, comprehensively concerning the ES factors, such as ES period, intensity, frequency, electrical pulse and interval change, was used to modify Rat pheochromocytoma PC-12 cells behaviours through a large size non-functional graphene nano-film (NGNF) which was prepared by spray coating the high conducting graphene sheets onto polyurethane film. The constant ES was used to select the optimized ES intensity and as well as control. The optimized ES condition with intensity of 100 mV/mm was shown to significantly enhance PC-12 cell differentiation, neurite extension and growth. Comparing the results by different ES, i.e. the constant 100 mV/mm and the programmed ES 100 mV/mm at 1 Hz and 10 Hz, the programmed ES increased longer neuritis length. The positive enhancement to nerve behaviour by programmed ES was still significant maintained at long periods of ES (48 h).