Morphology Of PC12 Cells Research Articles

Bu zhong Yiqi Decoction ameliorates mild cognitive impairment by improving mitochondrial oxidative stress damage via the SIRT3/MnSOD/OGG1 pathway

Ethnopharmacological relevanceBu-Zhong-Yi-Qi Decoction(BZYQD) is a traditional formula commonly used in China, known for its effects in tonifying Qi and raising Yang. It can relieve symptoms of cognitive impairment such as forgetfulness and lack of concentration caused by qi deficiency, which is common in aging and debilitating. However, much of the current research on BZYQD has been focused on its impact on the digestive system, leaving its molecular mechanisms in improving cognitive function largely unexplored. Aim of the studyCognitive decline in the aging central nervous system is intrinsically linked to oxidative damage. This study aims to investigate the therapeutic mechanism of BZYQD in treating mild cognitive impairment caused by qi deficiency, particularly through repair of mitochondrial oxidative damage. Materials and methodsA rat model of mild cognitive impairment (MCI) was established by administering reserpine subcutaneously for two weeks, followed by a two-week treatment with BZYQD/GBE. In vitro experiments were conducted to assess the effects of BZYQD on neuronal cells using a H2O2-induced oxidative damage model in PC12 cells. The open field test and the Morris water maze test evaluated the cognitive and learning memory abilities of the rats. HE staining and TEM were employed to observe morphological changes in the hippocampus and its mitochondria. Mitochondrial activity, ATP levels, and cellular viability were measured using assay kits. Protein expression in the SIRT3/MnSOD/OGG1 pathway was analyzed in tissues and cells through western blotting. Levels of 8-OH-dG in mitochondria extracted from tissues and cells were quantified using ELISA. Mitochondrial morphology in PC12 cells was visualized using Mito Red, and mitochondrial membrane potential was assessed using the JC-1 kit. ResultsBZYQD treatment significantly improved cognitive decline caused by reserpine in rats, as well as enhanced mitochondrial morphology and function in the hippocampus. Our findings indicate that BZYQD mitigates mtDNA oxidative damage in rats by modulating the SIRT3/MnSOD/OGG1 pathway. In PC12 cells, BZYQD reduced oxidative damage to mitochondria and mtDNA in H2O2-induced conditions and was associated with changes in the SIRT3/MnSOD/OGG1 pathway. ConclusionBZYQD effectively counteracts reserpine-induced mild cognitive impairment and ameliorates mitochondrial oxidative stress damage through the SIRT3/MnSOD/OGG1 pathway.

The structurally diverse phytocannabinoids cannabichromene, cannabigerol and cannabinol significantly inhibit amyloid β-evoked neurotoxicity and changes in cell morphology in PC12 cells.

Phytocannabinoids (pCBs) have been shown to inhibit the aggregation and neurotoxicity of the neurotoxic Alzheimer's disease protein beta amyloid (Aβ). We characterized the capacity of six pCBs-cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), cannabidivarin (CBDV), cannabidiol (CBD) and Δ9 -tetrahydrocannabinol (Δ9 -THC)-to disrupt Aβ aggregation and protect against Aβ-evoked neurotoxicity in PC12 cells. Neuroprotection against lipid peroxidation and Aβ-induced cytotoxicity was assessed using the MTT assay. Transmission electron microscopy was used to visualize pCB effects on Aβ aggregation and fluorescence microscopy, with morphometrics and principal component analysis to assess PC12 cell morphology. CBD inhibited lipid peroxidation with no significant effect on Aβ toxicity, whilst CBN, CBDV and CBG provided neuroprotection. CBC, CBG and CBN inhibited Aβ1-42 -induced neurotoxicity in PC12 cells, as did Δ9 -THC, CBD and CBDV. CBC, CBN and CBDV inhibited Aβ aggregation, whilst Δ9 -THC reduced aggregate density. Aβ1-42 induced morphological changes in PC12 cells, including a reduction in neuritic projections and rounded cell morphology. CBC and CBG inhibited this effect, whilst Δ9 -THC, CBD and CBDV did not alter Aβ1-42 effects on cell morphology. These findings highlight the neuroprotective activity of CBC, CBG and CBN as novel pCBs associated with variable effects on Aβ-evoked neurite damage and inhibition of amyloid β aggregation.

The Protective Effect of Docosahexaenoic Acid on PC12 Cells in Oxidative Stress Induced by H2O2 through the TrkB-Erk1/2-CREB Pathway

Docosahexaenoic acid (DHA) has attracted plenty of interest in the prevention of neurodegenerative diseases. Although the beneficial effects of DHA on the central nervous system function are recognized, more information on the molecular mechanisms involved in its neuroprotective effects is required. The present study aimed to evaluate the effects of DHA on the function of mitochondria, neurite growth-related proteins signaling pathway, and neural signal transmission. In this study, PC12 cells were treated with H2O2 (400 μM) to establish an oxidative damage model. Results showed that DHA improved the viability and morphology of PC12 cells. DHA significantly increased the antioxidant capacity, mitochondrial membrane potential, and activity of ATPase in the cells. Furthermore, the phosphorylation levels of tyrosine kinase receptor (BTrkB), phospholipase C-γ1 (PLCγ1), calcium/calmodulin-dependent protein kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), and cAMP-response element-binding protein (CREB) were upregulated by DHA. The damage on F-actin induced by H2O2 was reversed by DHA, indicating that DHA could protect neurite outgrowth. In addition, DHA increased the content of acetylcholine and γ-aminobutyric acid while decreasing glutamic acid. These results revealed that DHA could protect PC12 cells from damage induced by H2O2 through the TrkB-ERK1/2-CREB pathway.

Cell guidance on peptide micropatterned silk fibroin scaffolds

Guiding neuronal cell growth is desirable for neural tissue engineering but is very challenging. In this work, a self-assembling ultra-short surfactant-like peptide I3K which possesses positively charged lysine head groups, and hydrophobic isoleucine tails, was chosen to investigate its potential for guiding neuronal cell growth. The peptides were able to self-assemble into nanofibrous structures and interact strongly with silk fibroin (SF) scaffolds, providing a niche for neural cell attachment and proliferation. SF is an excellent biomaterial for tissue engineering. However neuronal cells, such as rat PC12 cells, showed poor attachment on pure regenerated SF (RSF) scaffold surfaces. Patterning of I3K peptide nanofibers on RSF surfaces significantly improved cellular attachment, cellular density, as well as morphology of PC12 cells. The live / dead assay confirmed that RSF and I3K have negligible cytotoxicity against PC12 cells. Atomic force microscopy (AFM) was used to image the topography and neurite formation of PC12 cells, where results revealed that self-assembled I3K nanofibers can support the formation of PC12 cell neurites. Immunolabelling also demonstrated that coating of I3K nanofibers onto the RSF surfaces not only increased the percentage of cells bearing neurites but also increased the average maximum neurite length. Therefore, the peptide I3K could be used as an alternative to poly-l-lysine for cell culture and tissue engineering applications. As micro-patterning of neural cells to guide neurite growth is important for developing nerve tissue engineering scaffolds, inkjet printing was used to pattern self-assembled I3K peptide nanofibers on RSF surfaces for directional control of PC12 cell growth. The results demonstrated that inkjet-printed peptide micro-patterns can effectively guide the cell alignment and organization on RSF scaffold surfaces, providing great potential for nerve regeneration applications.

Effect of ligustilide on oxygen and glucose deprivation/reperfusion-induced mitochondria fission in PC12 cells

This study aimed to investigate the effect and mechanism of ligustilide, the main active ingredient in Ligusticum wallichii, on mitochondria fission after PC12 cell injury induced by oxygen and glucose deprivation/reperfusion(OGD/R). In the experiment, an OGD/R model was established in vitro, and PC12 cells were pre-treated with ligustilide for 3 h, and then the cell viability was detected by CCK-8 method. The effect of different concentrations of ligustilide on the morphology of PC12 cells after OGD/R injury was observed under an inverted microscope. Transmission electron microscopy was used to observe the mitochondrial fission of PC12 cells after OGD/R injury. DCFH-DA immunofluorescence staining method was used to detect intracellular reactive oxygen species(ROS) changes. Changes in mitochondria membrane potential(MMP) were detected by flow cytometry. Hochest 33258 was used to observe the apoptosis of PC12 cells. Western blot was used to detect changes in cytochrome C(Cyt C) content in mitochondria and cytoplasm, and mitochondrial fission-related proteins Drp 1 and Fis 1. All results showed that compared with the model group, ligustilide significantly increased the survival rate of PC12 cells and the number of cells. Further experiments showed that ligustilide inhibited the release of ROS and decline of mitochondrial membrane potential in PC12 cells after OGD/R injury. Moreover, ligustilide reduced the release of Cyt C and promoted the expressions of Drp1 and Fis1 in mitochondrial fission proteins. Verification experiments showed that mitochondrial fission inhibitor mdivi-1 decreased cell survival rate and inhibited fission. The results indicated that ligustilide exerted neuro-protective effects by promoting mitochondrial fission and reducing cell damage. It preliminary proves that the mechanism of ligustilide on ischemic brain injury may be related to the promotion of mitochondrial fission and the maintenance of cell homeostasis.

Transfer of mitochondria from mesenchymal stem cells derived from induced pluripotent stem cells attenuates hypoxia-ischemia-induced mitochondrial dysfunction in PC12 cells.

Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury. Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology, the mechanisms are not fully understood. To address this issue, we first co-cultured 1.5 × 105 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1, and then intervened with cobalt chloride (CoCl2) for 24 hours. Reactive oxygen species in PC12 cells was measured by Mito-sox. Mitochondrial membrane potential (?Ψm) in PC12 cells was determined by JC-1 staining. Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining. Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy. Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry. Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria. Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl2 for 24 hours to detect adenosine triphosphate level in PC12 cells. CoCl2-induced PC12 cell damage was dose-dependent. Co-culture with mesenchymal stem cells significantly reduced apoptosis and restored ?Ψm in the injured PC12 cells under CoCl2 challenge. Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling, the disappearance of cristae, and chromatin margination in the injured PC12 cells. After direct co-culture, mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells. The transfer efficiency was greatly enhanced in the presence of CoCl2. More importantly, inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl2-induced PC12 cell injury. Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.

Effect of electrospinning parameters on morphological properties of PVDF nanofibrous scaffolds

Smart materials like piezoelectric polymers represent a new class of promising scaffold in neural tissue engineering. In the current study, the fabrication processing parameters of polyvinylidine fluoride (PVDF) nanofibrous scaffold are found as a potential scaffold with nanoscale morphology and microscale alignment. Electrospinning technique with the ability to mimic the structure and function of an extracellular matrix is a preferable method to customize the scaffold features. PVDF nanofibrous scaffolds were successfully fabricated by the electrospinning technique. The influence of PVDF solution concentration and other processing parameters like applied voltage, tip-to-collector distance, feeding rate, collector speed and the solvent were studied. The optimal parameters were 30 w/v% PVDF concentration, 15 kV applied voltage, 18 cm tip-to-collector distance, 0.5 ml/h feeding rate, 2500 rpm collector speed and N,N′-dimethylacetamide/acetone as a solvent. The mean fiber diameter of the obtained scaffold was 352.9 ± 24 nm with uniform and aligned morphology. Finally, the cell viability and morphology of PC-12 cells on the optimum scaffold indicated the potential of PVDF nanofibrous scaffold for neural tissue engineering.

The modification research of bionic silk fibroin nerve guidance conduits by silk sericin

Objective To explore the modification of bionic silk fibroin nerve conduits (SF-NCs) by silk sericin. Methods The innovative SS/SF blended-NCs was fabricated by a vertical sequential cooling thermal induced phase separation (TIPS) processing with SF solution added sericin in proportion, its morphology was observed by Scanning electron microscopy (SEM), X-ray diffraction (XRD) and infrared spectroscopy (FTIR) were used to detect its internal molecular structure. MTT assay was used to quantitatively analyzed the PC12 cells viability co-cultured with the innovative SS/SF-NCs, SEM was used to observe the adhesion and morphology of PC12 cells seeded into the innovative SS/SF, PC12 cells were used to assess the NGF bioactivity released from the SS/SF. Results The SEM results showed that the new fabricated SS/SF-NCs had linearly oriented lamellar-like multiple-channel which distributed evenly, got great changes on the channel microstructure and their mechanical properties had been greatly improved, compared to SF-NCs. The XRD and FTIR results showed that the SS/SF-NCs had the similar internal molecular structure with natural silk. The spaces between parallel lamellar-like channels, porosities and compressive strengths of the SS/SF-NCs decreased with decreasing Sequential freezing temperature. MTT assay results showed that the viability of PC12 cell was better than the control group (P < 0.05). The SEM observation indicated that PC12 cells showed good adhesion and differentiation with neuritis outgrowth during the period of co-culture with the SS/SF-NCs. NGF release from the innovative SS/SF-NCs was prolonged over 4 weeks, and remained bioactive. Conclusion The new fabricated SS/SF-NCs modified though silk sericin, which was highly bionic the structure of peripheral nerve fasciculus, had excellent mechanical properties and could be used as another alternative of artificial nerve conduits. Key words: Nerve guidance conduits (NCs); Silk sericin/silk fibroin (SS/SF) blended; Bionic; Modification; Peripheral nerve

Proteasome inhibitor-induced autophagy in PC12 cells overexpressing A53T mutant α-synuclein.

The aim of the present study was to examine the effects of proteasome inhibitor (PI)-induced autophagy on PC12 cells overexpressing A53T mutant α-synuclein (α-syn) by detecting alterations in the levels of microtubule-associated protein 1A/1B light chain (LC3)+ autophagosomes and the lysotracker-positive autolysosomes using immunofluorescence, the expression of LC3-II using western blot analysis and the morphology of PC12 cells using transmission electron microscopy. It was found that the addition of MG132 (500 nmol/l) significantly increased the number of autophagosomes and autolysosomes and upregulated the expression of LC3-II. The autophagy inhibitor 3-methyladenine (3-MA) completely inhibited the autophagy induced by MG132 (500 nmol/l). The autophagy enhancer trehalose significantly increased the number of autophagosomes and autolysosomes and improved the protein level of LC3-II induced by MG132. To examine the effect of PI-induced autophagy on the degradation of A53T mutant α-syn, the expression of α-syn was detected by western blot analysis. It was revealed that MG132 increased the expression of A53T α-syn and trehalose counteracted the increase of A53T α-syn induced by MG132. Combined inhibition of 3-MA and PI significantly increased the accumulation of A53T α-syn as compared with treatment using either single agent. In addition, combination of MG132 (500 nmol/l) with trehalose (50 mmol/l) or 3-MA (2 mmol/l) markedly decreased the cell viability as compared with treatment using either single agent individually as demonstrated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These results suggest that the PI, MG132, could induce autophagy in PC12 cells overexpressing A53T mutant α-syn and this autophagy could be completely inhibited by 3-MA, indicating that PI-induced autophagy is mediated by the upregulation of the macroautophagy class III PI3K pathway. PI-induced autophagy may act as a compensatory degradation system for degradation of A53T α-syn when the ubiquitin-proteasome system is impaired. Autophagy activation may directly contribute to the survival of PC12 cells treated with proteasome inhibitors. The present study may assist in illuminating the association between PI and autophagy in the pathogenesis of Parkinson’s disease.

The PERK-eIF2α signaling pathway is involved in TCDD-induced ER stress in PC12 cells

Studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces apoptotic cell death in neuronal cells. However, whether this is the result of endoplasmic reticulum (ER) stress-mediated apoptosis remains unknown. In this study, we determined whether ER stress plays a role in the TCDD-induced apoptosis of pheochromocytoma (PC12) cells and primary neurons. PC12 cells were exposed to different TCDD concentrations (1, 10, 100, 200, or 500nM) for varying lengths of time (1, 3, 6, 12, or 24h). TCDD concentrations much higher than 10nM (100, 200, or 500nM) markedly increased glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) levels, which are hallmarks of ER stress. We also evaluated the effects of TCDD on ER morphology in PC12 cells and primary neurons that were treated with different TCDD concentrations (1, 10, 50, or 200nM) for 24h. Ultrastructural ER alterations were observed with transmission electron microscopy in PC12 cells and primary neurons treated with high concentrations of TCDD. Furthermore, TCDD-induced ER stress significantly promoted the activation of the PKR-like ER kinase (PERK), a sensor for the unfolded protein response (UPR), and its downstream target eukaryotic translation initiation factor 2 α (eIF2α); in contrast, TCDD did not appear to affect inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), two other UPR sensors. Importantly, TCDD significantly inhibited eIF2α phosphorylation and triggered apoptosis in PC12 cells after 6–24h of treatment. Salubrinal, which activates the PERK-eIF2α pathway, significantly enhanced eIF2α phosphorylation in PC12 cells and attenuated the TCDD-induced cell death. In contrast, knocking down eIF2α using small interfering RNA markedly enhanced TCDD-induced cell death. Together, these results indicate that the PERK-eIF2α pathway plays an important role in TCDD-induced ER stress and apoptosis in PC12 cells.

THE FABRICATION AND CHARACTERIZATION OF LINEARLY ORIENTED LAMELLAR-LIKE MULTIPLE-CHANNEL SILK FIBROIN NERVE CONDUITS

Objective To explore how to fabricate a innovative silk fibroin nerve conduits (SF-NCs) with linearly oriented multiple-channel structure. Methods The innovative SF-NCs was fabricated by a vertical sequential cooling-thermal induced phase separation (TIPS) processing, its morphology was observed by Scanning electron microscopy (SEM), MTT assay was used to quantitatively analyzed the PC12 cells viability co-cultured with the innovative SF-NCs, SEM was used to observe the adhesion and morphology of PC12 cells seeded into the innovative SF-NCs, PC12 cells were used to assess the NGF bioactivity released from the SF-NCs. Results The SEM results showed that the new fabricated SF-NCs had linearly oriented lamellar-like multiple-channel, which distributed evenly. The spaces between parallel lamellar-like channels (ds), porosities and compressive strengths (σc, Ec) of the SF-NCs decreased with decreasing T ft . MTT assay results showed that the viability of PC12 cell was better than the control group (P &lt; 0.05). The SEM observation indicated that PC12 cells showed good adhesion and differentiation with neuritis outgrowth during the period of co-culture with the SF-NCs. NGF release from the innovative SF-NCs was prolonged over 4 weeks, and remained bioactive. Conclusion The new fabricated SF-NCs with special topography structure, excellent mechanical properties and good biocompatibility. Importantly, the SF-NCs are also biomimicking the anatomy structure of peripheral nerve fasciculus and could be used as another alternative of artificial nerve conduits.

Protective effects of Aloe vera extract on mitochondriaof neuronal cells and rat brain

To investigate the effects of Aloe vera extract (AV) on mitochondria in rat pheochromocytoma (PC12) cells and rat brain and to study the mechanism of its neuroprotection. After treatment, the morphology of PC12 cells was observed under microscope, the activity of mitochondria in PC12 cells was measured by MTT method, and the mitochondrial membrane potential (MMP) in PC12 cells was detected by JC-1 method. The mitochondrial function in rat brain was detected by resazurin method. The production of malondialdehyd (MDA) in rat brain mitochondria was tested by thiobarbaturic acid (TBA) assay. AV could improve mitochondrial damage induced by azide sodium (NaN3) in PC12 cells. The viability of PC12 cells treated with NaN364 mmol x L(-1) for 4 h decreased by 47.8%, and AV at 1 and 10 mg x L(-1) could respectively increase the viability of NaN3-treated cells by 16.7% (P < 0.05) and 22.3% (P < 0.01). MMP in PC12 cells in AV 1 and 10 mg x L(-1) group was significantly higher than that of NaN3-treated group (P < 0.05). AV also protected the structure and function of mitochondria in rat brain. AV at 10 mg x L(-1) had protective effect on mitochondria function impair induced by NaN3 (P < 0.01). AV 1 and 10 mg x L(-1) markedly inhibited the lipid peroxidation of brain mitochondria induced by Fe2+ -cysteine (P < 0.05, P < 0.01). AV has protective effects on mitochondria of neuronal cells and rat brain.

The protective effect of the insulin against paraquat-induced PC12 cells

Objective To observe dopamine (DA) D2R protein expression in the cultured PC 12 cells and the effect of insulin on the survival rate,morphology,and DA of paraquat (PQ)-induced PC12 cells. Methods Immunoprecipitate Western blotting method was performed to observe DA D2R protein expression in PC 12 cells,MTT assay was used to analyze the changes in viability and morphology of PC 12 ceils which were exposed to different concentrations of PQ and insulin. Results (1) DA D2R protein was expressed in PC 12 ceils. (2) Normal PCI2 cells bodies showed fusiform shape and the synapses were in-tegrity. The cells which were exposed to the 600 μmol/L PQ became ball-like, vacuolar degeneration oc-urred,and the synapse became shorter or disappeared. But the morphology of PC12 cells had a little difference between the normal PC12 and the insulin groups,except that the cells were fusiform shape or a-nomalism but not round shape,and the synapses grew. (3) With the increase of the concentration of PQ, the viability of the cells was decreased. Insulin increased the viability of the ceils which were exposed to 600 μmol/L PQ. Insulin elevated DA concentration both in the normal PC12 cells and those exposed to 600 μmol/L PQ,but there was no significant difference ( P > 0.05 ). Conclusion Insulin could protect the PC12 dopaminergic neurons from injury induced by PQ. Key words: Parkinson's disease; Paraquat; Insulin; PC12 cells

Sciatic nerve conditioned medium depleted of pro-NGF modulates sodium currents and neurite outgrowth in PC12 cells

Excitability and axon/dendrite specification are the most distinctive features in the establishment of neuronal polarization. Conditioned medium from rat sciatic nerve (CM) induced a neuronal-like morphology in PC12 cells. Here we show that CM neuritogenic activity is limited to the induction of dendrites in PC12 cells. However, treatment of these cells with CM in combination with a generic inhibitor for tyrosine kinase receptors (k252a) promoted the enhancement of neurite length, development of axons and induction of sodium currents. On the other hand, specific inhibition of TrkA and p75 NTR receptors in CM-treated cells reduced the neurite length in comparison with cells treated only with CM, although the effect over the induction of sodium currents was continuously observed. These results suggested that CM had some components that, even though are able to start the morphological cell differentiation and produce short neurites (likely acting through TrkA and p75 NTR), can restrain further neurite extension. Depletion of pro-NGF isoforms from CM produced a similar effect as the exerted by k252a, TrkA and p75 NTR receptor inhibitors in CM-treated cells, inducing the elicitation of sodium currents. These results suggested that the effect of CM might be mediated through pro-NGF. The difference between the results obtained with the generic inhibitor for Trk receptors and the specific inhibitors for TrkA and p75 NTR receptors in CM-treated cells, suggested that alternative pathways could be used to regulate neurite elongation, axon specification and sodium currents in PC12 cells. These findings represent important clues to improve the understanding of the initiation of neuronal polarity.

Protective Effects of Salidroside on Hypoxia/Reoxygenation Injury by Sodium Hydrosulfite in PC12 Cells

Hypoxia/reoxygenation causes cellular injury and death associated with many pathophysiologic conditions, including respiratory disorders, myocardial ischemia, and tumour progression diseases. Neuronal pheochromocytoma (PC12) cells are widely used as a model system for neurologic research and are subject to chemical hypoxia induced with sodium hydrosulfite (Na2S2O4), a common oxygen-consuming agent. Salidroside, which is the main active component of the famous traditional Chinese herb Rhodiola rosea. L. (Crassulaceae), has been proved to possess many bioactivities. In this article, we studied the protective effects of salidroside on hypoxia/reoxygenation injury in PC12 cells induced by Na2S2O4. Cultures of PC12 cells were exposed for 1 h to 10 mM Na2S2O4 for hypoxia, followed by reoxygenation for 2 h. The results showed that salidroside was very stable in medium and was not harmful to PC12 cells at the experimental concentrations of 0 ∼ 200 µg/mL. The cytoprotection by salidroside was dose-dependent, and the cell viability was 41.8 ± 5.7%, 62.4 ± 4.1%, and 92.2 ± 3.7% at 0, 50, and 100 µg/mL of salidroside, respectively. The level of released LDH significantly decreased from 513.5 ± 5.5% (without salidroside) to 258.1 ± 6.3% (with 100 µg/mL salidroside). Flow cytometry was performed to measure apoptotic rate. The results of flow cytometry assay indicated that the apoptotic rate was 17.0 ± 1.2% after hypoxia/reoxygenation injury. When the cells were treated with salidroside 12.5, 50 and 100 µg/mL, the apoptotic rate was 9.5 ± 0.9%, 7.4 ± 0.5%, and 4.5 ± 0.4%, respectively. In addition, our results were confirmed by inspection of cell morphology of PC12 cells. Treatment with salidroside (12.5, 50, 100 µg/mL) significantly prevented the cells from morphologic changes. All the above results showed salidroside could effectively protect PC12 cell against hypoxia/reoxygenation injury.

The Ras-GRF1 exchange factor coordinates activation of H-Ras and Rac1 to control neuronal morphology.

The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRFdeltaN, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRFdeltaC, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRFdeltaC binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size.

Lhx9andLhx9α: Differential Biochemical Properties and Effects on Neuronal Differentiation

The Lhx9 LIM-homeodomain transcription factor and its truncated isoform Lhx9alpha are generated by alternative splicing of the Lhx9 gene. Here we investigated the differential functional properties of these two isoforms. Lhx9alpha, which lacks parts of the homeodomain, was unable to bind DNA in EMSA experiments, but was able to associate with CLIM cofactors in GST pull-down assays. In transfection experiments in PC12 cells, Lhx9alpha fusion constructs systematically showed a nuclear localization, as opposed to Lhx9 fusion constructs, which also localized to the cytoplasm. Moreover, Lhx9 increased NGF-induced neuronal differentiation of PC12 cells. Lhx9alpha, on the other hand, did not significantly increase neuronal differentiation but had an effect on the morphology of PC12 cells. Finally, as tested by RT-PCR experiments on transfected PC12 cells, Lhx9 was not able to induce the transcription of Lhx9alpha. Our results show significantly different functional properties for Lhx9 and Lhx9alpha, and suggest that Lhx9alpha can compete away limiting amounts of nuclear CLIM cofactors. Thus, Lhx9 and Lhx9alpha isoforms could be implicated in regulating various aspects of neuronal differentiation.

Caspase activation independent of cell death is required for proper cell dispersal and correct morphology in PC12 cells

Caspase activation is indispensable for the proper execution of apoptosis. However, to date, little is known about other possible physiologic functions for this class of enzymes in addition to their well-defined role in apoptosis. In this report, we described an action of caspase-3 involving cell dispersion that is independent of cell death. Using an in vitro neuronal model system consisting of PC12 cells, we observed a transient activation of caspase-3 both in situ and by Western blot analysis that was evident at 1 h following plating, was maximal by 3 h, and was attenuated by 24 h. Preincubation of PC12 cells with either the caspase-3 inhibitor, DEVD, or antisense caspase-3 oligonucleotides caused cells to be more rounded in appearance and led to a failure of cells to disperse properly. Additional experiments demonstrated a possible target for caspase cleavage to be the cytoskeletal protein, tau. These data suggest a requirement for caspase activation and subsequent disassembly of the cytoskeleton during cell dispersion and represent a novel role for caspases that may allow for proper migration of neurons to target locations during development.

Nimodipine and flunarizine have different effects on survival and morphology of PC12 cells during nerve growth factor deprivation

The purpose of this study was to examine the effect of antagonists of different subtypes of Ca 2+ channels (nimodipine and flunarizine) and two types of Ca 2+ chelating agents (the cell permeant Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethylester (BAPTA-AM) and the cell non-permeant Ca 2+ chelator EGTA) on neurite retraction and cell death of nerve growth factor (NGF)-differentiated PC12 cells after NGF deprivation. We demonstrated that flunarizine and nimodipine, but not BAPTA-AM and EGTA, provided protection against cell death due to NGF deprivation. Using time-lapse videomicroscopy and quantitative image analysis, we found that retraction of neurites was an early and fast phenomenon after removal of NGF. None of the compounds tested (flunarizine, nimodipine, BAPTA-AM, EGTA) could prevent the retraction of neurites.

In VitroStudies of Acrylamide Neurotoxicity in Rat Pheochromocytoma (PC12) Cells

This review discusses our studies on molecular mechanisms of acrylamide neurotoxicity by using the rat pheochromocytoma (PC12) cell line. The results showed that: a) acrylamide altered the gross morphology of PC12 cells; b) acrylamide induced neurofilament accumulation in PC12 cells; c) the effects of acrylamide on PC12 cells are consistent with its neurotoxicity in vivo; d) acrylamide stimulated neurofilament protein synthesis in PC12 cells; e) acrylamide did not act via nerve growth factor (NGF) receptor gp140trk to regulate neurofilament synthesis in PC12 cells; f) dexamethasone antagonised NGF and/or acrylamide-induced neurofilament protein synthesis and expression; and g) acrylamide differentially regulated the mRNA levels of three neurofilament subunit genes in PC12 cells. These molecular studies provide the first evidence that: a) there are distinctive and convergent signalling pathways for NGF-regulated and acrylamide-regulated neurofilament expression; b) acrylamide may differentially regulate the expression of each subunit, resulting in aberrant accumulation of neurofilament proteins; and c) there is a dexamethasone-sensitive signalling step common to NGF and acrylamide. These results could partially explain the mechanisms of neurofilament accumulation in distal axonal swellings, a pathognomonic feature of acrylamide neurotoxicity.