Non-differentiated PC12 Cells Research Articles

In Vitro Neurotoxicity Resulting from Exposure of Cultured Neural Cells to Several Types of Nanoparticles.

Laboratory and industrial production of various nanoparticles, single-walled nanotubes (SWNTs), fullerene (C60), cadmium selenide (CdSe) quantum dots, carbon black (CB), and dye-doped silica nanospheres (NSs), has greatly increased in the past 15 years. However, little research has been done to analyze the toxicity of these materials. With recent studies showing that nano-substances can cross the blood–brain barrier, we examined the neurotoxicity of these manufactured nanoparticles. By employing the rat PC-12 neuronal-like cell line as the basis for our studies, we were able to evaluate the toxicity caused by these five nanoparticles. The level of toxicity was measured by testing for cell viability using the lactate dehydrogenase (LDH) cell viability assay, morphological analysis of changes in cellular structures, and Western blot analyses of αII-spectrin breakdown products (SBDP) as cell death indicators. Our results showed cytotoxicity in nondifferentiated PC-12 cells exposed to CB (10–100 µg/mL), SWNTs (10–100 µg/mL), C60 (100 µg/mL), CdSe (10 µg/mL), CB (500 µg/mL), and dye-doped silicon NSs (10 µg/mL). Exposure to higher concentrations (100 µg/mL) of SWNTs, CB, and C60 increased the formation of SBDP150/145, as well as cell membrane contraction and the formation of cytosolic vacuoles. The incorporations of the nanoparticles into cell cytoplasm were observed using the fluorescent dye-doped NSs in both nondifferentiated and nerve growth factor (NGF)-differentiated PC-12 cells. When PC-12 cells are differentiated, they appeared to be even more sensitive to cytotoxicity of nanoparticles such as CB 10 nm (10–100 µg/mL), CB 100 nm (10–100 µg/mL), and CdSe (1–10 µg/mL).

On the role of protein disulfide isomerase in the retrograde cell transport of secreted phospholipases A2.

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A2 (sPLA2) in snake venom, binds specifically to protein disulfide isomerase (PDI) in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA2. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b’. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA2s have the same fold as Atx. The first three sPLA2s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA2 in the Atx—hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA2s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA2 and hPDI is however different from that of the other sPLA2s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA2s confirmed the model-based expectation that GV sPLA2 was a more effective inhibitor than GX sPLA2, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA2s by assisting the retrograde transport of these molecules from the cell surface. The sPLA2–hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA2s in relation to their action intracellularly.

An RNA synthesis inhibition assay for detecting toxic substances using click chemistry

Biological risk assessment studies of chemical substances that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication, but also with transcription. There is no simple method for the detection of the DNA lesion-induced inhibition of transcription. Here, we report an assay for estimating the toxicity of chemical substances by visualizing transcription in mammalian cells using nucleotide analog 5-ethynyluridine (EU) and its click chemistry reaction. Ultraviolet light and representative chemical substances (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) of DNA- damaging agents show toxicity, as indicated by RNA synthesis inhibition in response to DNA damage in HeLa cells. Using titanium dioxide, we observed RNA synthesis inhibition in response to the rutile form, but not the anatase form, indicating that rutile titanium dioxide is a toxic substance. Because this method is based on the transcriptional response to DNA lesions, we can use terminally differentiated neuron-like PC12 cells, the differentiation of which can be induced by nerve growth factors, for evaluating chemical substances. Ultraviolet light and some chemicals (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) inhibited RNA synthesis in non-differentiated PC12 cells. Conversely, camptothecin and cisplatin did not inhibit RNA synthesis in differentiated PC12 cells, but 4-nitroquinoline-1-oxide, mitomycin C, and etoposide did. And using titanium dioxide, we did not observed any RNA synthesis inhibition. These data suggest that this method might be used to estimate the potential risk of chemical substances in differentiated mammalian cells, which are the most common cell type found in the human body.

156. Immune-cell products from mice with an autoimmune disease affect apoptosis, proliferation and metabolism of PC12 cells, a model of sympathetic neurons

Splenic sympathetic innervation decreases with the progression of the autoimmune, lymphoproliferative disease that Fas-deficientlpr/lpr mice develop, an alteration that contributes to its pathogenesis. We studied whether this derangement could be related to the hyperactive lpr/lpr immune system, and whether immune-derived products can influence the viability of sympathetic neurons. For this purpose, non-differentiated PC12 cells, an in vitro model of sympathetic neurons, were treated with supernatants from cultures of splenocytes from 11- and 40-week-old male and female C57BL/6 J (wild-type) and C57BL/6 J lpr/lpr mice. Immune-derived products from both lpr/lpr and wild-type mice induced apoptosis, but also supported proliferation, altered dopamine production and decreased the metabolic activity of PC12 cells. However, supernatants derived fromlpr/lpr immune cells induced more marked changes in all these parameters than those obtained from wild-type mice. Furthermore, supernatants obtained from lpr/lpr mice at more advanced stages of the disease appear to be more powerful in influencing the mentioned parameters than those derived from younger animals in which the disease was still not fully manifested. In contrast to the lpr/lpr-derived supernatants, the capacity of supernatants from lymphoid cells of wild-type mice to support PC12 proliferation seemed to predominate over its pro-apoptotic effect. These results strengthen the possibility that immune-derived products contribute to the alterations in splenic sympathetic innervation observed during the development of the autoimmune disease in lpr/lpr mice.

Apoptosis induced clustering of IP3R1 in nuclei of non-differentiated PC12 cells

Inositol 1,4,5-trisphosphate (IP(3)) receptors are emerging as key sites for regulation by pro- and anti-apoptotic factors. Induction of apoptosis for 3 h increased mRNA and protein levels of type 1 IP(3) receptors in non-differentiated (ND), but not in differentiated (D) PC12 cells. Inhibitors of the IP(3) R's calcium release-2-aminoethoxydiphenyl borate (2-APB) and xestospongin-completely prevented Bax and caspase-3 mRNA increase after treatment with the apoptosis inducer set (AIK), and this reinforces the importance of IP(3) R1 in the apoptosis of ND PC12 cells. Apoptosis induction not only increases the IP(3) R1 protein, but it also causes formation of IP(3) R1 clusters in the nucleus which most likely result from fusion of the nucleoplasmic reticulum and/or IP(3) R1 translocation to the nucleus. This is quite similar to the observations noted after overexpression of IP(3) R1 in PC12 cells. The amount of IP(3) induced calcium release was higher in control than in AIK-treated cells. From our results we propose that after the apoptosis induction the amount of intranuclear calcium decreased dramatically due to the increase of calcium permeability of the nuclear calcium store vesicles. Therefore, increase of the calcium permeability may result from IP(3) receptors translocation to nuclei that can boost the calcium transport through IP(3) receptors.

Gene expression pattern in PC12 cells with reduced PMCA2 or PMCA3 isoform: selective up-regulation of calmodulin and neuromodulin

Cellular calcium homeostasis is controlled predominantly by the plasma membrane calcium pump (PMCA). From four PMCA isoforms, PMCA1 and PMCA4 are ubiquitous, while PMCA2 and PMCA3 are found in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in non-differentiated PC12 cells changed the cell morphology and triggered neuritogenesis. Using the microarrays, real-time PCR and immunodetection, we analyzed the effect of PMCA2 or PMCA3 reduction in PC12 cells on gene expression, with emphasis on calmodulin (CaM), neuromodulin (GAP43) and MAP kinases. In PMCA-suppressed lines total CaM increased, and the calm I and calm II genes appeared to be responsible for this effect. mRNA and protein levels of GAP43 were increased, however, the amount of phosphorylated form was lower than in control cells. Localization of CaM/GAP43 and CaM/pGAP43 differed between control and PMCA-reduced cells. In both PMCA-modified lines, amounts of ERK1/2 increased. While pERK1 decreased, the pERK2 level was similar in all examined lines. PMCA suppression did not change the p38 amount, but the p-p38 diminished. JNK2 protein decreased in both PMCA-reduced cells without changes in pJNK level. Microarray analysis revealed distinct expression patterns of certain genes involved in the regulation of cell cycle, proliferation, migration, differentiation, apoptosis and cell signaling. Suppression of neuron-specific PMCA isoforms affected the phenotype of PC12 cells enabling adaptation to the sustained increase in cytosolic Ca(2+) concentration. This is the first report showing function of PMCA2 and PMCA3 isoforms in the regulation of signaling pathways in PC12 cells.

Role of collapsin response mediator protein-2 in neurite outgrowth of PC12 cells

Collapsin response mediator proteins (CRMPs) are involved in cell differentiation and axonal growth. In this study, we investigated neurite outgrowth and the expression of CRMP-2 in PC12 cells. Nondifferentiated PC12 cells hardly express CRMP-2, but the expression of CRMP-2 increases with neurite outgrowth. We established a stable CRMP-2-overexpressing PC12 cell line and found that PC12 cells, which constitutively overexpress CRMP-2, were unable to extend neurites after stimulation with the nerve growth factor or the neural cell adhesion molecule, a procedure that promotes neurite outgrowth in untransfected cells. In contrast, a PC12 cell line deficient in CRMP-2 extends neurites after the stimulation of nerve growth factor or neural cell adhesion molecule.

Regulation of Kalirin by Cdk5

Kalirin, one of the few Rho guanine nucleotide exchange factors (GEFs) that contains spectrin-like repeats, plays a critical role in axon extension and maintenance of dendritic spines. PC12 cells were used to determine whether Cdk5, a critical participant in both processes, regulates the action of Kalirin. Expression of Kalirin-7 in nondifferentiated PC12 cells caused GEF-activity-dependent extension of broad cytoplasmic protrusions; coexpression of dominant-negative Cdk5 largely eliminated this response. The spectrin-like repeat region of Kalirin plays an essential role in this response, which is not mimicked by the GEF domain alone. Thr1590, which follows the first GEF domain of Kalirin, is the only Cdk5 phosphorylation site in Kalirin-7. Although mutant Kalirin-7 with Ala1590 retains GEF activity, it is unable to cause extension of protrusions. Kalirin-7 with an Asp1590 mutation has slightly increased GEF activity and dominant-negative Cdk5 fails to block its ability to cause extension of protrusions. Phosphorylation of Thr1590 causes a slight increase in GEF activity and Kalirin-7 solubility. Dendritic spines formed by cortical neurons in response to the expression of Kalirin-7 with Ala1590 differ in shape from those formed in response to wild-type Kalirin-7 or Kalirin-7 containing Asp1590. The presence of Thr1590 in each major Kalirin isoform would allow Cdk5 to regulate Kalirin function throughout development.

FXYD6 Is a Novel Regulator of Na,K-ATPase Expressed in the Inner Ear

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.

NOX1/NADPH oxidase negatively regulates nerve growth factor-induced neurite outgrowth

Reactive oxygen species produced by NADPH oxidase are involved in the neuronal death associated with various neurodegenerative disorders. However, the role of NADPH oxidase in neuronal differentiation has not been well characterized. In nondifferentiated PC12 cells, the mRNA level of NOX1, a catalytic subunit of NADPH oxidase expressed in nonphagocytes, was approximately 10 times higher than that of the phagocyte type subunit, NOX2 (gp91 phox), while the transcript of another isoform, NOX4, was not detected. Following nerve growth factor (NGF)-induced neurite outgrowth, the mRNA level of NOX1 and NOX2 was progressively increased and decreased, respectively. The NGF-induced increase in NOX1 mRNA was mediated by TrkA and accompanied by increased intracellular superoxide, which was suppressed by NADPH oxidase inhibitors. Unexpectedly, these inhibitors and superoxide scavengers significantly enhanced NGF-induced neurite outgrowth. Enhanced neurite outgrowth was similarly demonstrated in cells depleted with the NOX1 transcript by stable expression of ribozymes targeted for the NOX1 mRNA sequence. Furthermore, NGF-induced expression of βIII-tubulin was significantly augmented in cells treated with NADPH oxidase inhibitors or stably expressing ribozymes. Phosphatidylinositol-3 (PI3) kinase inhibitors, without affecting NGF-induced NOX1 expression, augmented NGF-induced neurite outgrowth but not in clones expressing ribozymes. Taken together, increased superoxide production by up-regulation of NOX1 may negatively regulate neuronal differentiation by suppressing excessive neurite outgrowth.

Cloning and characterization of the chick NMDA receptor subunit-1 gene

The N-methyl- d-aspartate family of glutamate receptors (NMDARs) are tetrameric cation channels including NR1, NR2, and possibly NR3 subunits. The physiological properties of the receptor are directly related to the subunit composition of the oligomer. Whereas NR1 is essential for the formation of functional channels, NR2 and NR3 play a modulatory role. This work reports, for the first time, the cloning of a non-mammalian NR1 gene, including the 5′-regulatory region. The chick gene spans 31 kb of genomic DNA sequence composed of 22 exons interrupted by 21 introns. The exon/intron organization and the deduced amino acid sequence up to the end of exon 19 showed 85% homology to mammalian NR1 cloned genes. Significant differences from mammals were found at the C-terminal region which in the chick gene, includes a novel exon (exon 20) previously identified at the mRNA level in the chick retina. The basal promoter activity was shown to reside within the proximal 377 bp of 5′-regulatory region. The transcriptional activity of the 5′-flanking region of the chick NR1 gene was shown to be higher in neuronally-differentiated PC12 cells and in chick retinal neurons, than in non-differentiated PC12 cells and Müller glia. Comparison of the 5′-flanking region of chick NR1 gene with mammalian NR1 genes suggests that, in spite of significant differences in the nucleotide sequence, they share common DNA binding sites such as RE1, SP1, AP2, CREB, NFκB, and MEF2; therefore, some of the molecular mechanisms involved in transcriptional regulation of NR1 gene expression could be conserved among species.

Differentiation intensifies the susceptibility of pheochromocytoma cells to antisense oligodeoxynucleotide-dependent suppression of acetylcholinesterase activity.

To investigate the effect of neuronal differentiation on the capacity of antisense oligonucleotides (AS-ODNs) to suppress the production of acetylcholinesterase (AChE) in rat pheochromocytoma cells, we tested seven 3'-phosphorothioated AS-ODNs targeted to ACHEmRNA and two control ODNs. Three different administration protocols were used: oligonucleotides were added at 1 microM for 24 hours to nondifferentiated PC12 cells, together with nerve growth factor (NGF) or 24 hours following NGF-induced cholinergic differentiation. The content of free thiol groups in lysed cells was measured to evaluate cell number, therefore, survival, and the rate of acetylthiocholine hydrolysis was the measure of AChE activity. Among nondifferentiated cells, over 95% survived treatment with 8 of 9 of the ODNs. Moreover, two AS-ODN suppressed AChE activity in non-differentiated PC12 cells by 16%-20% as compared with 10% suppression by control ODNs (P < or = 0.01). When added concurrently with NGF, one other AS-ODN suppressed AChE activity significantly better (28%) than the control ODNs (16%). Moreover, when added following NGF treatment, which induced a significant increase in AChE activity, four different AS-ODNs but not the control ODNs suppressed 20%-35% of the enhanced AChE activity (p < or = 0.01). Reduced levels of AChE mRNA but no difference in actin mRNA levels were observed by following the kinetics of RT-PCR amplification in differentiated PC12 cells treated with these four AS-ODNs, as compared with control cells. Our findings demonstrate a differentiation-related increase in the susceptibility of PC12 cells to inhibition by specific AS-ODNs, suggesting the use of this model system to select AS-ODNs for suppression of AChE levels in the treatment of neurodegenerative diseases associated with cholinergic malfunction.

Modulation of β-Amyloid Precursor Protein Secretion in Differentiated and Nondifferentiated Cells

The regulation of β-amyloid precursor protein (β-APP) secretion was compared in differentiated and nondifferentiated PC12 and B104 cells. Phorbol esters stimulated the release of β-APP in all cells examined. However, differentiated PC12 cells were much more sensitive to phorbol ester treatment than nondifferentiated PC12 cells and their β-APP release was also induced by the protein phosphatase inhibitor okadaic acid and the Ca++-ionophore A23187. In contrast, β-APP release from B104 cells was strongly stimulated by A23187 and to lesser degree by phorbol esters. This effect was most pronounced in nondifferentiated B104 cells, which might be due to a higher basic release of β-APP from differentiated B104 cells. Thus, the regulation of β-APP cleavage and release varies depending on the cell type and differentiation state of the cell.

The effect of α-latrotoxin on the neurosecretory PC12 cells differentiated by treatment with nerve growth factor

Neurosecretory PC12 cells differentiated in vitro by prolonged (at least 2 weeks) treatment with nerve growth factor were exposed to α-latrotoxin and studied by morphological and biochemical techniques. Cell monolayers or suspensions responded to the toxin with a prompt and massive release of neurotransmitter. The dose dependency ( K m ~ 5 × 10 −10 M) and the maximum release effect (~60% of the stored [ 3H]dopamine released within 8 min) were not appreciably different from the values found in non-differentiated PC12 cells. Moreover, the concentration dependency of the release was found to correspond closely to that of the [ 125I]α-latrotoxin binding to its specific sites (the α-latrotoxin receptors). The number of these receptors was over two-fold higher in differentiated than in undifferentiated cells. Since, however, differentiation implies a large increase in cell size and surface area, the receptor density (number/unit area) remained virtually unchanged. By radioautography the α-latrotoxin receptors were found to remain diffusely distributed at the entire surface of differentiated cells even when these were allowed to form synapses with myotubes. This situation is at variance with that demonstrated recently at the frog neuromuscular junction, where α-latrotoxin receptors are exclusively localized at the nerve terminal plasmalemma. Scanning and transmission electron microscopy revealed that the enlargements of neuntes where dense granules are preferentially accumulated—the varicosities and terminals—underwent swelling and extensive disorganization within a few minutes after the application of α-latrotoxin, whereas the cell bodies and the tracts of neurites occupied primarily by micro tubules were less severely affected. The greater sensitivity of varicosities and terminals with respect to the other parts of the differentiated cells, rather than the consequence of a specific addressing of the toxin to these structures, might be due to their vulnerability by toxin-induced events, such as the uncontrolled activation of ion fluxes.