Effects of Selenium and Topiramate on Cytosolic Ca2+ Influx and Oxidative Stress in Neuronal PC12 Cells

It has been widely suggested that selenium (Se) deficiency play an important role in the pathophysiology of epilepsy. It has been reported that Se provides protection against the neuronal damage in patients and animals with epilepsy by restoring the antioxidant defense mechanism. The neuroprotective effects of topiramate (TPM) have been reported in several studies but the putative mechanism of action remains elusive. We investigated effects of Se and TPM in neuronal PC12 cell by evaluating Ca²⁺ mobilization, lipid peroxidation and antioxidant levels. PC12 cells were divided into eight groups namely control, TPM, Se, H₂O₂, TPM + H₂O₂, Se + H₂O₂, Se + TPM and Se + TPM + H₂O₂. The toxic doses and times of H₂O₂, TPM and Se were determined by cell viability assay which is used to evaluate cell viability. Cells were incubated with 0.01 mM TPM for 5 h and 500 nM Se for 10 h. Then, the cells were exposed to 0.1 mM H₂O₂ for 10 h before analysis. The cells in all groups except control, TPM and Se were exposed to H₂O₂ for 15 min before analysis. Cytosolic Ca²⁺ release and lipid peroxidation levels were higher in H₂O₂ group than in control, Se and TPM combination groups although their levels were decreased by incubation of Se and TPM combination. However, there is no difference on Ca²⁺ release in TPM group. Glutathione peroxidase activity, reduced glutathione and vitamin C levels in the cells were lower in H₂O₂ group than in control, Se and TPM groups although their values were higher in the cells incubated with Se and TPM groups than in H₂O₂ groups. In conclusion, these results indicate that Se induced protective effects on oxidative stress in PC12 cells by modulating cytosolic Ca²⁺ influx and antioxidant levels. TPM modulated also lipid peroxidation and glutathione and vitamin C concentrations in the cell system.     

Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

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 (400 µM). 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 100 µM SNP pre-treatment in a murine macrophage cell line. The apoptosis caused by toxic-dose SNP treatment (400 µM) 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 100 µM 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 100 µM 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.     

Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q < 0.05; enrichment range 1.40–9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting β-cells and neurons and underline the existence of trans?nosology pathways in diabetes and its co-morbidities.     

Activation of Rho-Dependent Cell Spreading and Focal Adhesion Biogenesis by the v-Crk Adaptor Protein

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160^ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160^ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, α₃-integrin, and a higher-molecular-weight form of β₁-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.     

Binding-induced activation of overexpressed p185HER2 is essential in triggering neuronal differentiation of PC12 cells

To determine whether p185^HER2 overexpression per se triggers p185^HER2 cellular signaling or whether an extracellular signal is required, we transfected PC12 cells with the human erbB-2 proto-oncogene, and established a cell line that overexpresses p185^HER2. PC12-HER2 cells, maintained in suspension culture or plated on a collagen layer, showed the same morphology and growth rate as PC12 and PC12 mock-transfected control cells. When treated with monoclonal antibody (MAb) MGr6 or other anti-p185^HER2 MAbs, PC12–HER2 cells specifically underwent neuronal differentiation comparable to that induced by nerve growth factor (NGF), and the differentiation-inducing effect of the MAb was dramatically enhanced by the addition of a second anti-mouse IgG. MAb-induced cell differentiation correlated with p185^HER2 phosphorylation, recruitment of Shc and Grb-2 transducer molecules into complexes, and MAPK phosphorylation. These data indicate the requirement for a specific binding-induced activation of the overexpressed p185^HER2 receptor in inducing PC12 cell differentiation. PC12-HER2 cells represent a suitable system for selection of p185^HER2-activating ligands (peptides, phage-displayed peptides or proteins) or specific inhibitors of its tyrosine kinase activity. J. Cell. Biochem. 67:316–326, 1997. © 1997 Wiley-Liss, Inc.     

MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation

MicroRNAs (miRNAs) are small non-coding RNAs that control protein expression through translational inhibition or mRNA degradation. MiRNAs have been implicated in diverse biological processes such as development, proliferation, apoptosis and differentiation. Upon treatment with nerve growth factor (NGF), rat pheochromocytoma PC12 cells elicit neurite outgrowth and differentiate into neuron-like cells. NGF plays a critical role not only in neuronal differentiation but also in protection against apoptosis. In an attempt to identify NGF-regulated miRNAs in PC12 cells, we performed miRNA microarray analysis using total RNA harvested from cells treated with NGF. In response to NGF treatment, expression of 8 and 12 miRNAs were up- and down-regulated, respectively. Quantitative RT-PCR analysis of 11 out of 20 miRNAs verified increased expression of miR-181a^∗, miR-221 and miR-326, and decreased expression of miR-106b^∗, miR-126, miR-139-3p, miR-143, miR-210 and miR-532-3p after NGF treatment, among which miR-221 was drastically up-regulated. Functional annotation analysis of potential target genes of 7 out of 9 miRNAs excluding the passenger strands (*) revealed that NGF may regulate expression of various genes by controlling miRNA expression, including those whose functions and processes are known to be related to NGF. Overexpression of miR-221 induced neuronal differentiation of PC12 cells in the absence of NGF treatment, and also enhanced neuronal differentiation caused by low-dose NGF. Furthermore, miR-221 potentiated formation of neurite network, which was associated with increased expression of synapsin I, a marker for synapse formation. More importantly, knockdown of miR-221 expression by antagomir attenuated NGF-mediated neuronal differentiation. Finally, miR-221 decreased expression of Foxo3a and Apaf-1, both of which are known to be involved in apoptosis in PC12 cells. Our results suggest that miR-221 plays a critical role in neuronal differentiation as well as protection against apoptosis in PC12 cells.     

Presence of DNase γ-Like Endonuclease in Nuclei of Neuronal Differentiated PC12 Cells

DNase , which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase -like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca²⁺ and Mg²⁺, and is sensitive to Zn²⁺. The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase -like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.     

Zonisamide Attenuates MPP(+)-Induced Oxidative Toxicity Through Modulation of Ca2+ Signaling and Caspase-3 Activity in Neuronal PC12 Cells

Parkinson’s disease is an incurable progressive neurological condition caused by a degeneration of dopamine-producing cells characterized by motor and non-motor symptoms. The major mechanisms of the antiepileptic actions of ZNS are inhibition of voltage-gated Na⁺ channel, T-type voltage-sensitive Ca²⁺ channel, Ca²⁺-induced Ca²⁺ releasing system, and neuronal depolarization-induced glutamate release; and enhancement of release of inhibitory neurotransmitters; however, the detailed mechanism of antiparkinsonian effects of ZNS remains to be clarified. We aimed to investigate to the effect of ZNS on the oxidative stress, cell viability, Ca²⁺ signaling, and caspase activity that induced by the MPP⁺ model of Parkinson’s in neuronal PC12 cells. Neuronal PC12 cells were divided into four groups namely, control, ZNS, MPP⁺, and ZNS+MPP⁺ groups. The dose and duration of ZNS and MPP⁺ were determined according to cell viability (MTT) analysis which used to assess the cell viability. The cells in ZNS, MPP⁺, and ZNS+MPP⁺ groups were incubated for 5 h with 100 μM ZNS, 10 h with 100 μM MPP⁺, and 10 h with ZNS and MPP⁺, respectively. Lipid peroxidation and cytosolic free Ca²⁺ concentrations were higher in the MPP⁺ group than in control although their levels were lower in ZNS and the ZNS+MPP⁺ groups than in control. Reduced glutathione and glutathione peroxidase values were lower in the MPP⁺ group although they were higher in the ZNS and the ZNS+MPP⁺ groups than in control. Caspase-3 activity was lower in the ZNS group than in the MPP⁺ group. In conclusion, ZNS induced modulator effects on the oxidative stress, intracellular Ca²⁺, and the caspase-3 values in an experimental model of Parkinson disease.     

Effect of culture container and carbohydrate content on in vitro slow growth storage of the cherry rootstock ‘Gisela<Superscript>®</Superscript>5’

The present study investigated the effects of gas-tight and gas-permeable culture containers and different sucrose concentrations, as well as sucrose and mannitol combinations on the development of an effective in vitro slow growth storage protocol (at 4 °C, in darkness) for ‘Gisela^®5’ shoot cultures. ‘Gisela^®5’ is the most widely used cherry rootstock in Europe. This dwarf triploid hybrid has many advantages over the conventional cherry rootstocks. Optimizations for the cold storage of ‘Gisela^®5’ in vitro shoot cultures included use of storage medium supplemented with 10, 20, 30, 45, and 60 g L^?1 sucrose and sucrose (15, 30 g L^?1) and mannitol (15 g L^?1) combinations, contained in gas-tight glass jars and gas-permeable ‘Star Pac?’ bags. Cold storage was prolonged to 12 months, during which in every 3 months, cultures were evaluated. Possibility of 16 month-cold storage in gas-tight glass jars was also explored, during which gas chromatographic analysis was performed for the detection of CO₂ and ethylene accumulation for the first 5 months of cold storage. Our results showed that both the 12- and 16-month conservations were possible, especially when 45 or 60 g L^?1 sucrose was supplemented to storage medium, contained in glass jars. Mannitol inclusion to the storage medium was also effective to reduce the metabolic activity of the shoot cultures during storage; however, it did not have a significant positive influence on shoot quality in post-conservation.