A quantitative bioassay for nerve growth factor,using PC12 clones expressing different levels of trkA receptors

Nerve growth factor (NGF) is a neurotrophin required for differentiation, development, and survival of the sympathetic nervous system, with many of its biological effects being mediated via trkA receptors. There is a need for a standard quantitative bioassay for NGF, to be used in basic research and in pharmaceutical studies. The objective of the present research was to develop a selective, quantitative, and reliable bioassay for NGF, using a morphological criterion: neurite cell outgrowth. In addition, we aimed to apply the aforementioned bioassay to measure NGF administered to mice. Pheochromocytoma PC12 cell variants including wild-type cultures, and a trkA-overexpressing stable transfectant PC12-6.24-I, PC12nnr5, and PC12EN lacking trkA receptors, were used. Dose-response curves were generated with NGF β-subunit (2.5S) purified from mouse submaxillary glands. Our results demonstrated that the bioassay was sensitive to 0.3–20 ng/mL, and selective, as neurite outgrowth was not seen by any other growth factor other than NGF. In addition, variant clones PC12nnr5 and PC12EN, lacking trkA receptors, did not respond to NGF. The bioassay detected NGF in serum of mice injected with NGF. This novel developed bioassay can serve as a model system for various neuroscience purposes.     

Fucosyl-GM1 expression and amyloid-beta protein accumulation in PC12 cells

Gangliosides, sialic acid-containing glycosphingolipids, are ubiquitously expressed in all eukaryotic cells and are localized primarily in the plasma membrane. For a rat pheochromocytoma cell line, PC12, which has been used frequently as a model for investigating events leading to neuronal differentiation, it is generally thought that GM1 is a major ganglioside, based on reactivity with the probe cholera toxin B subunit (Ctxb). From a series of biochemical studies, however, it has been reported that no GM1 is expressed in PC12 cells. In this study, we have reevaluated GM1 expression and Ctxb reactivity in PC12 cells and a subcloned line, PC12D cells. Flow cytometric analysis with Ctxb revealed that about 30-50% of PC12 cells were reactive with Ctxb. However, a detailed biochemical analysis showed that PC12 cells express abundantly a different ganglioside, fucosyl-GM1, instead of GM1, and the reactivity of Ctxb in the PC12 cells actually arose from its interaction with fucosyl-GM1, which also interacts with this ligand. Because it has been claimed that amyloid-beta protein (Abeta) interacts with GM1 in PC12 cells to provide "seeding" for amyloid to accumulate, we further evaluated this possibility and found that Abeta is mostly likely interacting with fucosyl-GM1 in this cell line. Our data thus suggest that a specific interaction may occur between Abeta and fucosyl-GM1 for the accumulation of amyloid in PC12 cells.     

Development of an ischemic tolerance model in a PC12 cell line.

Although ischemic tolerance has been described in a variety of primary cell culture systems, no similar in vitro models have been reported with any cell line. A model of ischemic preconditioning in the rat pheochromocytoma PC12 cell line is described here. When compared to nonpreconditioned cells, preexposure of PC12 cells to 6 hours of oxygen and glucose deprivation (OGD) significantly increased cell viability after 15 hours of OGD 24 hours later. Flow cytometry analysis of cells labeled with specific markers for apoptosis, Annexin V, and Hoechst 33342, and of DNA content, revealed that apoptosis is involved in OGD-induced PC12 cell death and that preconditioning of the cells mainly counteracts the effect of apoptosis. Immunocytochemistry of caspase-3, a central executioner in the apoptotic process, further confirmed the activation of apoptotic pathways in OGD-induced PC12 cell death. This model may be useful to investigate the cellular mechanisms involved in neuronal transient tolerance following ischemia.     

Transmitter, ion channel and receptor properties of pheochromocytoma (PC12) cells: a model for neurotoxicological studies.

The increased desire to use in vitro techniques in neurotoxicology has resulted in the search for clonal cell lines which may be useful for studying disruption by neurotoxicants of various aspects of neuronal physiology and biochemistry. One such cell line is the PC12 cell, a clonal cell line derived from a pheochromocytoma of the rat adrenal medulla. When cultured under normal conditions, PC12 cells resemble adrenal chromaffin cells in morphology, physiology and biochemistry. However, when cultured in the presence of nerve growth factor (NGF) or several other compounds, PC12 cells differentiate to resemble sympathetic neurons morphologically and functionally. Differentiation and the resultant physiological and biochemical changes are some of the most attractive and useful features of this cell line. PC12 cells release, depending on the conditions, dopamine, norepinephrine and acetylcholine and contain Na, K and Ca channels and other membrane receptors, including receptors coupled to G-proteins. Moreover, the relative proportion of various subtypes of Ca channels changes during differentiation. Thus, PC12 cells provide an excellent model for studying chemical disruption of processes associated with neuronal differentiation, synthesis, storage and release of neurotransmitters, function and regulation of ion channels and interactions of compounds with membrane bound receptors. The ability of PC12 cells to differentiate in response to NGF and other compounds allows for selective expression of certain channels and proteins and for comparisons of responses in undifferentiated and differentiated cells. The prominent neurotoxicant methylmercury causes potent reductions in uptake of 45Ca and binding of ligands associated with various subpopulations of Ca channels in the PC12 cells, as well as currents carried through putative Ca channels.     

NOGO—A在PC12细胞分化过程中的表达及意义

目的探讨NOGO—A在PC12细胞生长发育过程中的表达及意义。方法培养大鼠嗜铬细胞瘤细胞系PC12细胞，用神经生长因子诱导其分化，并于倒置显微镜下随机取20视野计数观察细胞增值和轴突生长情况。采用免疫荧光染色、逆转录酶聚合酶链反应（RT—PCR）及免疫印迹法等方法检测诱导后第1d、第3d、第5d、第7d PC12细胞中NOGO—AmRNA及蛋白的表达及变化，并留取细胞培养液检测多巴胺水平。结果未分化的PC12细胞中未检测到NOGO—A mRNA及蛋白表达。经神经生长因子诱导的PC12细胞，细胞轴突不断生长，NOGO—AmRNA及蛋白的表达逐渐增高（P〈0．05）。PC12细胞在分化过程中多巴胺（DA）分泌水平无明显差别。结论PC12细胞向交感神经元分化的过程中NOGO—A的表达逐渐增强，推测NOGO—A在神经元发育早期可能促进轴突生长。但对多巴胺激素释放的调节不明显。     

Functional Recovery in Hemiparkinsonian Primates Transplanted with Polymer-Encapsulated PC12 Cells

Cross-species neural grafting of cell lines immunoisolated by a permselective polymer membrane represents a promising source of neuroactive molecules for the treatment of neurodegenerative diseases. Utilization of a cell line of xenogeneic origin is advantageous since the transplanted cells will be rejected by the host immune system in case of breakage of the immunoisolating envelope. Polymer-encapsulated PC12 cells, a dopaminergic cell line derived from a rat pheochromocytoma, were transplanted in five Macaca fascicularis monkeys which had been previously rendered hemiparkinsonian by a unilateral carotid injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Well-preserved, tyrosine hydroxylase positive encapsulated PC12 cells were observed in the lesioned striatum for up to 5 months after implantation. Four out of five monkeys which received polymer-encapsulated PC12 cells showed significant behavioral improvement, whereas three monkeys implanted with either encapsulated bovine chromaffin cells or empty polymer envelopes showed no amelioration.     

Long-term cross-species brain transplantation of a polymer-encapsulated dopamine-secreting cell line

Cross-species transplantation of dopamine-releasing cell lines protected against immune rejection by a semi-permeable synthetic membrane may provide a source of neurotransmitters for the treatment of Parkinson's disease. Experiments were carried out to assess whether polymer-encapsulated PC12 cells, a catecholaminergic cell line derived from a rat pheochromocytoma, could survive in vitro as well as in vivo after implantation in the striatum of adult guinea pigs. When maintained in vitro, the encapsulated PC12 cells exhibited good survival, proliferated, and spontaneously released dopamine for at least 6 months. They also retained the capacity for depolarization-elicited dopamine release. In vivo, well-preserved tyrosine hydroxylase-positive PC12 cells were observed in capsules implanted for 4, 8, and 12 weeks. Unencapsulated PC12 cells or cells in nonintact capsules did not survive transplantation at any of these time periods. The survival of encapsulated PC12 cells transplanted across species suggests that polymer encapsulation may provide an alternative for xenotransplantation of secretory cells in the absence of systemic immunosuppression.     

Origin of the beta cells of the islets of Langerhans is further questioned by the expression of neuronal intermediate filament proteins, peripherin and NF-L, in the rat insulinoma RIN5F cell line.

Intermediate filament proteins of the rat insulinoma RIN5F cell line were characterized. Two-dimensional gel analysis followed by immunostaining of proteins demonstrated that these cells express both peripherin and the low-molecular-mass neurofilament protein (NF-L); this was confirmed for peripherin by immunohistochemistry, peptide analysis and Northern blot. No expression of these proteins could be detected with these same methods either in the adult pancreas or in the tumor at the origin of the cell line, although such expression was apparent on sections of rat pancreas at embryonal day 16. These results were compared to those obtained on the rat pheochromocytoma PC12 cell line: expression in the adrenal medulla of the embryo, no expression either in the adult tissue or in the tumor, but solely in the derived cell line. The expression of neuronal intermediate filament proteins in the rat insulinoma RIN5F cell line is discussed in relation to its similarity in the rat pheochromocytoma PC12 cell line, and its meaning as to the developmental cell lineage; an ectodermal origin is suggested for the pancreatic islet cells.     

Comparison of adrenal medullary, carotid body and PC12 cell grafts in 6-OHDA lesioned rats

The survival and functional properties of dispersed cell implants of catecholaminergic cells obtained from the peripheral nervous system of adult rats (adrenal medulla and carotid body glomus cells) and PC12 cells from a rat pheochromocytoma cell line were examined following transplantation into the striatum of the adult rat. The host animals, all with unilateral 6-hydroxydopamine (6-OHDA) nigrostriatal lesions, were divided into 5 groups: (1) PC12 cells transplanted into Cyclosporin-A treated hosts; (2) PC12 cell grafts into hosts without Cyclosporin-A treatment; (3) grafts of adrenal medullary cells; (4) grafts of glomus cells; and (5) vehicle controls. All animals were sacrificed one month after transplantation. Immunocytochemical staining for tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, was used to identify and characterize the grafted cells. PC12 cells were detected in four of six Cyclosporin-A treated rats, and two of these grafts developed into tumors. However, only one of the six non-Cyclosporin-A treated hosts was found to have surviving PC12 cells, and none of these rats developed tumors. No significant differences in rotational behavior were seen in either of the PC12 cell recipient groups. Grafted cells could be identified in all of the adrenal medullary and glomus cell recipients. However, the number of surviving cells was quite limited, with not more than 100 tyrosine hydroxylase-positive grafted cells found in any one recipient. Tyrosine hydroxylase-positive fibers were present adjacent to the transplants in these latter graft recipients, but the fibers appeared to be of host origin rather than from the grafts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reciprocal modulation of TrkA and p75NTR affinity states is mediated by direct receptor interactions

Equilibrium binding of ¹²⁵I-nerve growth factor (¹²⁵I-NGF) to cells coexpressing the tyrosine kinase receptor A (TrkA) and common neurotrophin receptor (p75^NTR), cells coexpressing both receptors where p75^NTR is occupied, and cells expressing only p75^NTR, revealed reciprocal modulation of receptor affinity states. Analysis of receptor affinity states in PC12 cells, PC12 cells in the presence of brain-derived neurotrophic factor (BDNF), and PC12^nnr5 cells suggested that liganded and unliganded p75^NTR induce a higher affinity state within TrkA, while TrkA induces a lower affinity state within p75^NTR. These data are consistent with receptor allosterism, and prompted a search for TrkA/p75^NTR complexes in the absence of NGF. Chemical crosslinking studies revealed high molecular weight receptor complexes that specifically bound ¹²⁵I-NGF, and were immunoprecipitated by antibodies to both receptors. The heteroreceptor complex of TrkA and p75^NTR alters conformation and/or dissociates in the presence of NGF, as indicated by the ability of low concentrations of NGF to prevent heteroreceptor crosslinking. These data suggest a new model of receptor interaction, whereby structural changes within a heteroreceptor complex are induced by ligand binding.     

Baculovirus p35 gene greatly enhances PC12 cell's resistance against oxidative stress

Oxidative stress is thought to be a major contributor to the progress of the Parkinson's Disease (PD) because of the high vulnerability of dopaminergic cells against oxidative stress. The present work demonstrates that with the expression of the baculovirus p35 gene, PC12 cells could gain a high resistance against oxidative toxicants, hydrogen peroxide (H(2)O(2)) and 6-hydroxydopamine (6-OHDA). The DNA fragmentation analysis showed that PC12 cells underwent apoptosis after exposure to H(2)O(2) or 6-OHDA, while PP35 cells, a p35-expressing PC12 cell line, did not. Flow cytometric analysis showed that treatment with 150 microM H(2)O(2) or 120 microM 6-OHDA for 24 h caused 52.86% or 66.36% apoptotic cell, respectively, in PC 12 cells, but only 4.26% or 5.80% in PP35 cells. The cell viability measured by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay indicated that H(2)O(2) and 6-OHDA induced a dose-dependent cell death on PC12 cells that were greatly remitted on PP35 cells. The viability of PP35 cells was even stronger than that of PC12 cells protected by glial cell line deprived neurotrophic factor (GDNF). The surviving PP35 cells remained normal cell morphology and showed positive with tyrosine hydroxylase (TH) immunocytochemical staining. These results indicate that baculovirus p35 gene possesses remarkable ability to rescue PC12 cells from death in experimental paradigms associated with oxidative stress.     

Viral Kirsten ras infection differentiates PC12 cells and enhances their survival upon implantation into brain

Neuronal cells from established cell lines can offer a well-characterized source of cells for transplantation to the brain that is an alternative to fetal neurons. The infection of members of the PC12 cell line with a retrovirus containing ras-oncogene leads to their neuronal differentiation without the need of nerve growth factor (NGF). We find that neoplastic, naive PC12 cells grafted to the striatum of normal adult rats cause the transient formation of large hemorrhagic cavities and do not survive. After differentiation by infection with Kirsten-ras murine sarcoma virus, and transplantation to the opposite striatum of the same brain, PC12 cells survive for at least 8 weeks and emit neurites. These neuron-like cells and their neurites retain tyrosine hydroxylase and choline acetyl transferase, as detected immunohistochemically. Thus, ras-primed PC12 cells may serve as a continuous source for both cholinergic and adrenergic transmitters, in vivo, without the need of exogenous nerve growth factor.     

Survival and differentiation within the adult mouse striatum of grafted rat pheochromocytoma cells (PC12) genetically modified to express recombinant beta-NGF.

Rat pheochromocytoma PC12 cells were genetically modified in vitro to express recombinant beta-nerve growth factor (beta-NGF) using a replication-deficient retroviral vector carrying the mouse beta-NGF gene and subsequently implanted into the striatum of a mouse model of Parkinson's disease. The fate of the genetically modified PC12 cells (PC12N.8) was assessed at varying times postimplantation by studying immunoreactivity (IR) to tyrosine hydroxylase (TH) or the rat NGF receptor (NGFR). In vitro, the genetically modified PC12 cells displayed a neuronal morphology in the absence of exogenous NGF which was characterized by extensive neurite outgrowth. In addition, the genetically modified PC12 displayed a catecholaminergic phenotype in vitro as assessed by TH-IR. Following implantation into the striatum, the survival of PC12N.8 cells was limited. Surviving cells could be identified by NGFR-IR, but not by TH-IR. In addition, PC12N.8 cells with a neuronal morphology similar to that observed in vitro were only rarely observed in vivo. No tumors were observed in PC12N.8 graft recipients up to 30 days postimplantation. In contrast, intrastriatal tumors were observed in 50% of the PC12 cell recipients. These data demonstrate that PC12 cells genetically modified in vitro to synthesize beta-NGF do not revert to the mitotic phenotype of the parent PC12 cell line following implantation into the adult striatum, an observation that suggests that these cells may continue to express recombinant beta-NGF in vivo. The data further suggest that the genetically modified PC12 cells lose the catecholaminergic phenotype following implantation into the striatal parenchyma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variant PC12 cell line that spontaneously differentiates and extends neuritic processes

The rat pheochromocytoma PC12 cells differentiate into neuronal-like cells in response to treatment with neurotrophins. The cells have been extensively used for investigating neuronal differentiation and axonal growth. Here we report the isolation of a variant PC12 cell line, named PC12-N1, which spontaneously differentiates and extends neuritic processes. The PC12-N1 cells expressed many neuronal specific proteins, including the synaptosomal associated protein of 25 kDa (SNAP-25), synaptotagmin, and synaptobrevin (also known as VAMP). The cells also expressed neurofilament protein of 68 kDa, a marker for differentiated neurons. In addition to the spontaneous neurite outgrowth, the PC12-N1 cells showed a marked increase in neurite outgrowth upon treatment with nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and cyclic AMP (cAMP). The activation of mitogen-activated protein (MAP) kinases was examined by immunoblot analysis using phospho-specific antibodies. No overactivation was observed with ERK1/2 or p38. However, the c-Jun N-terminal kinase JNK/SAPK was activated approximately 10-fold over the parental PC12 cells. These results suggest that activation of JNK/SAPK may be involved in the spontaneous neurite extension in the PC12-N1 cells. Moreover, the PC12-N1 cells may be used as a model for investigating molecular signaling mechanisms underlying neuronal differentiation and axonal outgrowth.     

Rottlerin通过抑制PKCδ磷酸化减轻6羟基多巴胺对多巴胺能细胞的毒性作用

目的观察蛋白激酶C6亚型（PKCδ）磷酸化在6羟基多巴胺（6-OHDA）引起的多巴胺能神经细胞死亡过程中的作用,探讨帕金森病中神经元缺失的分子机制。方法体外培养大鼠嗜铬细胞瘤细胞系PC12细胞,观察预先加入的PKC抑制剂（bisindolylmaleimide I,Go6976及Rottlerin）和激动剂佛波酯对6-OHDA毒性作用的影响,噻唑蓝比色法检测细胞存活率,免疫印迹法观察磷酸化PKC8的表达。结果PKC8抑制剂Rottlerin（2μmol／L）可抑制PKC8的磷酸化,减轻6-OHDA引起的细胞死亡,细胞存活率上升至69．6±2．63％（P〈0．05）。PKCα抑制剂bisindolylmaleimide I和钙依赖性PKC抑制剂Go6976对6-OHDA的毒性作用及PKCδ磷酸化均无显著影响,而PKC8激活剂佛波酯（100nmol／L）能提高PKC8磷酸化水平,加重6-OHDA的损害作用,使细胞存活率下降至单用6-OHDA组水平的49．8±5．06％（P〈0．001）。结论Rottlerin能抑制PKCδ的磷酸化,进而减轻6-OHDA对多巴胺能神经细胞死亡的诱导作用,说明PKCδ505位点丝氨酸的磷酸化是6-OHDA发挥毒性作用的关键,提示PKCδ在帕金森病病人神经元缺失中起重要作用。     

Pycnogenol protects neurons from amyloid-beta peptide-induced apoptosis

Neuronal apoptosis is one of the pathological features of Alzheimer's disease (AD). Morphological pathology reveals that neuronal apoptosis is associated with senile plaques containing amyloid-beta peptide (Abeta) in AD brains. Reactive oxygen species (ROS) has been proposed to be involved in the apoptotic mechanism of Abeta-mediated neurotoxicity. In the present study, using a rat pheochromocytoma (PC12) cell line, we investigated the effect of Pycnogenol (PYC), a potent antioxidant and ROS scavenger, on Abeta(25-35)-induced apoptosis and ROS generation. We used vitamin E, a known antioxidant agent, to verify the effect of PYC. Abeta(25-35)-induced apoptosis in PC12 cells was demonstrated by: (1) a dose-dependent loss of cell viability; (2) a time- and dose-dependent increase in the apoptotic cells; (3) an induction of DNA fragmentation; and (4) an increase in caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP). Our data showed that a significant increase in ROS formation preceded apoptotic events after PC12 cells were exposed to Abeta(25-35). We further found that PYC not only suppressed the generation of ROS but also attenuated caspase-3 activation, DNA fragmentation, PARP cleavage, and eventually protected against Abeta-induced apoptosis. Vitamin E also suppressed cell death and caspase-3 activation induced by Abeta(25-35). Taken together, these results suggest that ROS may be involved in Abeta-induced apoptosis in PC12 cells. They further suggest that PYC can reduce apoptosis, possibly by decreasing free radical generation in PC12 cells.     

Characterization of intermediate filaments in PC12 cells

A 57 kDa protein is the major polypeptide in intermediate filament (IF)-enriched cytoskeletal preparations obtained from the neuronal cell line PC12 (rat pheochromocytoma). Under the conditions used to assemble IF in vitro from other cultured cell lines, 10 nm filaments are formed after 2 cycles of disassembly-assembly from PC12 IF-enriched cytoskeletal preparations; the 57 kDa protein is the major component of the final IF pellet. The 57 kDa protein is immunologically related to the BHK-21 fibroblast 55 kDa protein (vimentin), but a comparison of the peptide maps of PC12 57 kDa and BHK 55 kDa indicates that they are different proteins. With the use of a polyclonal antiserum to the PC12 57 kDa protein, immunofluorescence observations of PC12 cells not treated with NGF reveal a juxtanuclear "knot"-like structure. After NGF treatment, the "knots" are less prominent and many IF arrays are seen coursing through the cytoplasm and extending into the neurites. These immunofluorescence observations of the distribution of IF are corroborated by fine-structural analyses. SDS-PAGE analyses indicate that IF-enriched cytoskeletons isolated from NGF-treated cells have a polypeptide composition similar to that of untreated cells, that is, the 57 kDa protein remains the major polypeptide. SDS-PAGE and immunoblotting analyses show that untreated and NGF-treated PC12 cells also contain relatively minor amounts of the 68, 150, and 200 kDa neurofilament triplet (NFT) proteins. Under immunofluorescence, only 5% of untreated PC12 cells are found to contain a juxtanuclear "knot" labeled with NFT antibodies, but with time following NGF treatment, the number of fluorescent cells increases. After about 2 weeks of NGF treatment, all of the PC12 cells appear to contain NFT antibody-positive filamentous structures. As assessed by immunofluorescence, the NFT polypeptides appear to codistribute with the 57 kDa protein in both untreated and NGF-treated PC12 cells. These data indicate that PC12 cells contain IF composed of a complex set of polypeptides, including a previously unidentified 57 kDa IF protein. While NGF may induce production of NFT polypeptides, there does not appear to be a "switch" from known mesenchymal IF polypeptide expression to NFT polypeptide expression upon stimulation of PC12 cells with NGF.     

Antioxidant effect of retinoic acid on PC12 rat pheochromocytoma.

Retinoic acid is a naturally occurring metabolite of vitamin A that influences the differentiation of a variety of neural cells in vitro. In the LA-N-1 human neuroblastoma line, retinoic acid treatment increases the binding of nerve growth factor (Bmax). The purpose of this study was to examine the effects of retinoic acid on PC12 rat pheochromocytoma, a neural crest-derived cell line that can be induced to express a sympathetic neuroblast-like phenotype by nerve growth factor treatment. In contrast to the differentiating effects of nerve growth factor, retinoic acid treatment of PC12 cells had a negligible effect on cellular morphology. However, treatment with retinoic acid enhanced the survival of PC12 cells following oxidative injury generated by H2O2 treatment in a manner that is qualitatively similar to that observed after nerve growth factor treatment. Also, there was an increase in 125I-nerve growth factor binding activity in solubilized PC12 membrane preparations derived from retinoic acid-treated PC12 cells. These data suggest that retinoic acid may play a role in neuronal development and in neuronal injury by stimulating the ability of neurons to cope with oxidative stress and/or by enhancing neuronal responsiveness to trophic factors such as the nerve growth factor.