Ruthenium red neurotoxicity and interaction with gangliosides in primary cortical cultures

Ruthenium red (RR) is an inorganic polycationic dye able to exert several effects on the nervous system, including neurodegeneration, both in vivo and in cell cultures. Gangliosides have been shown to protect cultured neurons against several damaging conditions, and it has been postulated that RR can interact with the negative charges of the sialic acid residues of these molecules. In the present work we have tested the effect of the trisialoganglioside GT1b and the monosialoganglioside GM1 on the RR-induced neuronal damage in primary cortical cultures, as well as on the binding of RR to synaptosomes. GT1b at 100–200 μM concentrations partially protected against RR-induced neurodegeneration, as judged by light microscopy and by measurement of the reduction of a tetrazolium salt, while GM1 was ineffective. GT1b, but not GM1, also partly blocked both RR binding and its diminution in the culture medium occurring during incubation. These results suggest that the three negative charges of GT1b enable it to interact with RR and as a consequence the entrance of the dye into the cells is blocked and neurotoxicity is diminished, although other mechanisms of protection cannot be excluded. Endogenous polysialic acid–containing molecules do not seem to be involved in RR effects, since the removal of sialic acid residues by treatment with neuraminidase did not prevent the cell damage. J. Neurosci. Res. 49:72–79, 1997. © 1997 Wiley-Liss Inc.     

Cooperative regulation of neurotensin content in PC12 pheochromocytoma cell cultures: effects of nerve growth factor, dexamethasone, and activators of adenylate cyclase

Nerve growth factor, dexamethasone, and forskolin or cholera toxin (CT) act cooperatively to increase the content of neurotensin (NT) in PC12 pheochromocytoma cells. Relatively small increases in NT content occur in the presence of NGF alone or dexamethasone alone, but not of forskolin or CT alone. Increases of 10- to 100-fold occur in the presence of NGF plus dexamethasone or NGF plus forskolin, and up to 600-fold increases occur in the presence of all three agents. These increases are extremely stable and persist for at least 2 weeks after removal of dexamethasone or forskolin. The complex regulation of NT stores in PC12 cells might reflect mechanisms that regulate NT content of normal chromaffin cells and/or neurons during development or in adult life. A small amount of stored NT is released in response to stimulation with 52 mM K+. This release is blocked in the presence of 2 mM Co2+, suggesting that it occurs via Ca2+-mediated exocytosis.     

Regulation of ionic currents in pheochromocytoma cells by nerve growth factor and dexamethasone

Growth factors and hormones induce differentiation of clonal pheochromocytoma (PC12) cells, which are derived from rat adrenal medulla chromaffin cells. On application of nerve growth factor (NGF), PC12 cells extend neurites and express properties characteristic of autonomic ganglion cells. In contrast, incubation of PC12 cells with a corticosteroid, dexamethasone (DEX), does not induce neurite formation but causes an increase in tyrosine hydroxylase activity, suggesting that the cells become chromaffin cell-like. The ability of NGF and DEX to regulate ionic currents has been less well studied. Therefore, we examined how long-term NGF and DEX treatments affected voltage-dependent Na, Ca, and K currents in PC12 cells. Voltage-dependent Na currents were observed only in a small fraction of the PC12 cells in the absence of NGF or DEX. Virtually all NGF-treated cells expressed Na currents within 7 d. DEX increased the number of cells expressing voltage-dependent Na current slowly over 3 weeks, but, unlike NGF, DEX did not change Na current density. Both NGF and DEX also affected the expression of voltage-dependent Ca currents. Most of the untreated cells had only sustained, high-threshold voltage-dependent Ca currents. Chronic application of NGF or DEX increased the fractions of the cells that showed transient, low-threshold T-type Ca currents in addition to the high-threshold currents. The T-type Ca current density, however, increased significantly only in NGF-treated cells. Neither DEX nor NGF affected the voltage-dependent K currents. These results suggest that the expression of voltage-dependent Na and Ca currents are differentially regulated by NGF and DEX. The distinction between treated and untreated cells decreased after 3 weeks in culture as older untreated cells showed increases in the fraction of cells expressing both Na and low-threshold Ca currents. A PC12 subline selected for adherence to uncoated plastic also showed increased fraction of cells expressing these currents, suggesting that interactions with substrate may also influence ionic current expression.     

Tumor necrosis factor-alpha and nerve growth factor synergistically induce iNOS in pheochromocytoma cells

Inducible nitric oxide synthase (iNOS) has been reported in tangle-bearing neurons of patients with Alzheimer's disease (AD), and can be induced by tumor necrosis factor-alpha (TNFalpha). High CNS levels of TNFalpha are associated with neurodegenerative diseases such as AD, where neurons dependent on neurotrophins such as nerve growth factor (NGF) are particularly affected. In this study we determined the effect of TNFalpha on iNOS in NGF-responsive pheochromocytoma (PC12) cells. We found that while TNFalpha and NGF alone were unable to induce iNOS, their simultaneous addition resulted in iNOS induction and the release of nitric oxide. Our results suggest that synergistic iNOS induction by TNFalpha and NGF may occur in selective population of NGF-responsive neurons in the presence of elevated CNS levels of TNFalpha.     

Cholesterol biosynthesis and the pro-apoptotic effects of the p75 nerve growth factor receptor in PC12 pheochromocytoma cells

Neocarzinostatin (NCS), an enediyne antimitotic agent, induces cell death in both p75NTR neurotrophin receptor (NTR)-positive and p75NTR-negative PC12 cells in a concentration-dependent fashion. However, p75NTR-positive cells demonstrate a higher susceptibility to NCS-induced cell damage. Furthermore, treatment of p75NTR-positive cells with the p75NTR-specific ligand, MC192, resulted in apoptosis, while treatment of these cells with the TrkA-specific ligand, NGF-mAbNGF30, protected them from NCS-induced death, implying that both the naked and liganded p75NTR receptors have a pro-apoptotic effect on PC12 cells. Microarray studies aimed at examining differential gene expression between p75NTR-positive and p75NTR-negative cells suggested that enzymes of the cholesterol biosynthetic pathway are differentially expressed. We therefore tested the hypothesis that altered cholesterol biosynthesis contributes directly to the pro-apoptotic effects of p75NTR in this PC12 cell-NCS model. Subsequent Northern blotting studies confirmed that the expression of p75NTR is associated with the upregulation of cholesterol biosynthetic enzymes including 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), farnesyl-diphosphate synthase, and 7-dehydro-cholesterol reductase. Mevastatin, an HMG CoA reductase inhibitor, converts the apoptosis susceptibility of p75NTR-positive cells to that of p75NTR-negative cells. It does so at concentrations that do not themselves alter cell survival. These studies provide evidence that the pro-apoptotic effects of p75NTR in PC12 cells are related to the upregulation of cholesterol biosynthetic enzymes and consequent increased cholesterol biosynthesis.     

脑缺血再灌注损伤中前体神经生长因子和成熟体神经生长因子及其受体的表达

实验观察脑缺血再灌注损伤中,前体神经生长因子和成熟体神经生长因子在缺血区皮质和非缺血区皮质的表达。结果发现在缺血区和非缺血区皮质中,前体神经生长因子表达在细胞外和胞浆,成熟体神经生长因子极低少量表达在细胞外。缺血侧皮质前体神经生长因子表达显著下降,至1d时达到最低值,成熟体神经生长因子在4h时表达增加,至3d时达到最低值,神经生长因子受体(p75NTR)在缺血后表达明显降低,至3d时达到峰值。证实脑缺血再灌注损伤的病理过程中：①前体神经生长因子是神经生长因子存在的主要形式。②脑缺血再灌注损伤影响了前体神经生长因子向成熟体神经生长因子的转化过程。③ 前体神经生长因子/p75NTR可能参与了缺血后的再灌注损伤。     

Herpes simplex virus replication in pheochromocytoma cell line that responds to nerve growth factor

Summary Cultured cells of neural origin (PC-12, pheochromocytoma cell line) respond to nerve growth factor (NGF) by extending neurites. These cells whether treated with NGF or not can be infected with herpes simplex virus and produce progeny virus. Viral antigens are detected on the cell surface and fusion of cells to form polykaryocytes takes place. Nucleocapsids are found within the cell nucleus and enveloped virus in present both in the cytoplasm and extracellular space. Virus was not observed within the neurites but alterations in the neurite microtubular structure occurred after infection.     

Nerve growth factor effects and receptors in cultured human neuroblastoma cell lines

We studied the effects of nerve growth factor (NGF) to determine whether neuroblastoma (NB) cells share the pattern of altered response to growth regulatory factors shown by various malignant transformed cells. NGF induces neurite outgrowth, arrests growth, and enhances survival in normal neurons and in the rat pheochromocytoma, a tumor cell closely related to NB. With respect to neurite outgrowth, lines SK-N-SH, SH-SY5Y, LA-N-5, and CHP-126 were sensitive, IMR-32 was resistant, and SH-EP1, SK-N-MC, MC-IXC, CHP-100, and CHP-134 were unresponsive. Conditioned media from unresponsive cells did not inhibit response in sensitive cells. Unexpectedly, NGF neither reduced the growth rate nor enhanced survival in any NB cell line. Conditioned medium from all NB cell lines enhanced 125I-NGF binding in embryonic sensory cells. Regulation of growth rate and neurite outgrowth, then, are separable. A fundamental defect in NB may be the acquisition of a capacity for growth and survival independent of NGF. 125I-NGF was bound to both Fast and Slow receptors in MC-IXC cells, but only to Slow receptors in NGF-responsive SH-SY5Y and LA-N-5 cells, showing Fast receptors are not required for neurite outgrowth. Independence from NGF-regulated growth and survival is unexplainable by an absence of NGF receptors.     

Fast and slow nerve growth factor binding sites in human neuroblastoma and rat pheochromocytoma cell lines: relationship of sites to each other and to neurite formation

We studied (a) the distribution and properties of fast and slow 125I-nerve growth factor (125I-NGF) binding sites in cultured human neuroblastoma (NB) cell lines that were categorized as responsive (N+) or unresponsive (N-) to NGF by neurite outgrowth, (b) whether fast or slow sites mediate actions of NGF, and (c) whether NGF-mediated conversion of fast to slow sites occurs in human NB and pheochromocytoma PC 12 cells. In human NB SH-SY5Y cells, the slow sites were trypsin resistant and binding was of high affinity. Loss of binding to the slow sites had a half-time of 25 to 30 min at 37 degrees C and was very slow at 4 degrees C. In contrast, the fast sites were trypsin sensitive and binding was of lower affinity; its dissociation half-time was less than 1 min at 4 degrees C and 37 degrees C. The association rate constants of both sites were about 0.8 to 1.2 X 10(7) M-1 sec-1. Some human NB cells had both fast and slow sites. The N+ human NB lines SH-SY5Y and LA-N-5 had only slow sites. Despite the virtual elimination of fast sites by trypsin in NB MC-IXC cells, remaining slow sites could still efficiently bind 125I-NGF. These observations showed that fast sites are not required for slow site binding, neurite outgrowth, or other demonstrated actions of NGF in some NB cells. In PC 12 cells, 125I-NGF initially bound to fast sites was not directly transferred to slow sites as required for NGF-mediated conversion. The association rate constants of fast and slow sites in PC12 cells were both about 2 X 10(7) M-1 sec-1. The association kinetics were consistent with simple bimolecular reactions in both NB and PC12 cells. The combined evidence in NB and PC12 cells did not support the hypothesis of NGF-mediated conversion of fast to slow sites.     

Serum vulnerability and time-dependent stabilization of neurites induced by nerve growth factor in PC12 pheochromocytoma cells

Cultures of PC12 pheochromocytoma cells were established on a polyornithine substratum in medium supplemented with the chemically defined N1 mixture in the presence or absence of Nerve Growth Factor (NGF). Normal cell proliferation in the absence of NGF was equally competent when fetal calf serum (FCS) was replaced with N1-supplemented medium. The differentiation of PC12 cells, which occurs upon NGF treatment, ultimately results in cell death without the addition of 0.1% FCS to the N1-supplemented medium. The combination of N1, 0.1% FCS, and NGF permits the PC12 cells to develop a neuritic outgrowth much earlier than when higher (1-10%) FCS levels are used. Neurite retraction is caused in a dose-dependent manner by a delayed presentation of FCS. Within 2 days of serum presentation, however, neurites regrow to achieve that percentage of neurite-bearing cells which is seen without a serum challenge. Moreover, the retraction response becomes less pronounced with time over the 8-day culture period for any given serum concentration. Among the N1 ingredients, only insulin and transferrin are needed by PC12 cells for survival whether in the dividing state or not. Neurite growth was not dependent on any of the N1 components.     

Characteristics of the association of nerve growth factor with primary cultures of rat sympathetic neurons

Long-term primary cultures of rat sympathetic neurons require NGF for survival and development. The kinetics of the interaction of 125I-NGF with sympathetic neuron cultures suggests the presence of diffusional barriers preventing a determination of true dissociation and association rate constants. Although the observed rate constants do not accurately reflect the microscopic interaction of NGF with receptor, the ratio of the observed rate constants does provide a good estimate of the KD. This value (1 X 10(-9)M) agrees with earlier steady state measurements of the KD. The association of 125I-NGF with neuronal cultures is temperature-dependent with internalization and retrograde transport occurring at 37 degrees C. The retrograde transport of 125I-NGF in compartmentalized neuronal cultures is concentration dependent and saturates at about 100 ng/ml (4 X 10(-9)M). The amount of 125I-NGF accumulated by retrograde transport appears to be increased subsequent to a period of NGF-starvation. The increase in uptake does not appear to be due to an increase in NGF receptor number since the number of binding sites is not greatly increased upon NGF starvation.     

Effects of insulin, insulin-like growth factor-II and nerve growth factor on neurite outgrowth in cultured human neuroblastoma cells

The identification of biologically important and chemically well-defined substances that can promote axon and dendrite formation would improve present understanding of the development of the nervous system. Physiological concentrations of insulin and insulin-like growth factor-II (IGF-II) reversibly enhanced neurite outgrowth (NTO) in human neuroblastoma SH-SY5Y cells cultured in media with and without serum. Nerve growth factor (NGF), in contrast, did not enhance NTO in serum-free media. Furthermore, anti-NGF antiserum inhibited NGF but not insulin-enhanced NTO. Insulin increased [3H]leucine and [3H]uridine uptake. These increases, together with increased NTO, were inhibited by cycloheximide and actinomycin D, respectively. The inhibition of NTO by cycloheximide was reversible. Human neuroblastoma cell lines that were responsive by NTO to NGF were also responsive to insulin, with the exception of line CHP-270. Moreover, cell lines unresponsive by NTO to NGF, and to tumor promoters, were uniformly unresponsive to insulin. These findings suggest that there are common defects in distal sites, because specific NGF and tumor promotor receptors are present in these lines. Insulin increased [3H]thymidine uptake in SH-SY5Y and CHP-100 cells. However, the enhancement of NTO by insulin and IGF-II in SH-SY5Y cells was independent of the cellular proliferation rate. Our results, together with the observations of others, suggest that insulin and IGF-II may modulate NTO in the nervous system.     

Effect of nerve growth factor on C-1300 murine neuroblastoma tumor growth and catecholamine content in neonatally sympathectomized mice

The in situ C-1300 murine neuroblastoma (MNB) tumor model was used to investigate the influence of exogenously administered nerve growth factor (NGF) on tumor growth and tissue catecholamine concentration in mice sympathectomized with 6-hydroxy-dopamine (6-OHDA) on postnatal days 4-10. Mice were implanted with 1 x 10(6) disaggregated MNB cells 3 days after termination of 6-OHDA administration. NGF (12-15 micrograms/mouse/day) treatment was initiated at the time of MNB cell implantation and continued until sacrifice of the animal. The time interval between tumor cell implantation and detection of palpable tumor (tumor onset time), transverse tumor diameter, tumor weight, tumor weight to body weight ratio, and tumor catecholamine concentration were determined. Neonatal sympathectomy caused a decrease in myocardial norepinephrine concentration of 88% compared with vehicle-treated animals as well as a significant reduction in total body and organ weight. Average body, brain, heart, and spleen weights were decreased 31%, 16%, 25%, and 42%, respectively, below control values. The daily injection of NGF, from the time of MNB tumor implantation to sacrifice, did not prevent these effects of chemical sympathectomy from being expressed. Tumor onset time following implantation of MNB cells was significantly increased in neonatally sympathectomized mice and was not altered by treatment with NGF. In contrast, the decrease in MNB tumor growth rate observed in sympathectomized mice was reversed by administration of NGF. Mean tumor weight and mean tumor to body weight ratio were 89% and 115% of comparable control values, respectively, in sympathectomized mice receiving exogenous NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroprotective effects of ultrasound-guided nerve growth factor injections after sciatic nerve injury

Nerve growth factor (NGF) plays an important role in promoting neuroregeneration after peripheral nerve injury. However, its effects are limited by its short half-life; it is therefore important to identify an effective mode of administration. High-frequency ultrasound (HFU) is increasingly used in the clinic for high-resolution visualization of tissues, and has been proposed as a method for identifying and evaluating peripheral nerve damage after injury. In addition, HFU is widely used for guiding needle placement when administering drugs to a specific site. We hypothesized that HFU guiding would optimize the neuroprotective effects of NGF on sciatic nerve injury in the rabbit. We performed behavioral, ultrasound, electrophysiological, histological, and immunohistochemical evaluation of HFU-guided NGF injections administered immediately after injury, or 14 days later, and compared this mode of administration with intramuscular NGF injections. Across all assessments, HFU-guided NGF injections gave consistently better outcomes than intramuscular NGF injections administered immediately or 14 days after injury, with immediate treatment also yielding better structural and functional results than when the treatment was delayed by 14 days. Our findings indicate that NGF should be administered as early as possible after peripheral nerve injury, and highlight the striking neuroprotective effects of HFU-guided NGF injections on peripheral nerve injury compared with intramuscular administration.     

Regulation of nerve growth factor gene expression in primary brain monolayer cultures: effects of dibutyryl cyclic AMP and sodium butyrate

Primary brain cultures composed of a heterogeneous population of cells, most of which reacted with anti-fibronectin and only few with anti-GFAP antiserum, were found to synthesize a mRNA indistinguishable from mouse submandibular gland nerve growth factor (NGF) mRNA in hybridization characteristics and electrophoretic behavior in agarose gels. The levels of this mRNA were increased 2.5-10 fold by 1 mM dibutyryl cyclic AMP (db-cAMP) and sodium butyrate within 24 h, but not by 10 microM forskolin, and were unaffected by serum in the culture medium. These observations demonstrate that brain cells synthesize a NGF mRNA in primary culture and that the butyrate moiety of db-cAMP enhances NGF gene expression in these cells, probably by a modification of chromatin structure in and around the NGF gene.     

Identification of nerve growth factor receptors in primary cultures of chick neural crest cells

Primary cultures of chick neural crest cells obtained from explanted neural tubes have binding sites for radioiodinated nerve growth factor ([125I]NGF) but not for radioiodinated epidermal growth factor ([125I]EGF). The binding of [125I]NGF was shown to be a specific and saturable process with a high affinity (Kd = 0.3 nM) for the ligand. Despite the expression of these NGF binding sites, incubation of the neural crest cultures with nerve growth factor did not induce neurite outgrowth; no morphological alterations were observed. This was not due to an inability of the cells to express a neuronal phenotype, since the neural crest cells spontaneously differentiated into neurite-bearing cells. However, the nerve growth factor binding sites do appear to be functional receptors, since nerve growth factor could produce a modest induction of ornithine decarboxylase. The quantity of nerve growth factor binding sites seemed to be independent of the phenotype expressed by the neural crest cells, since both pigmented cells and neuron-like neural crest cells exhibited binding. These findings suggest that the differentiation of neural crest cells into mature nerve growth factor-responsive neurons may involve the coupling of nerve growth factor receptors to cellular responses important in the expression of the neuronal phenotype.     

Regulation of brain-derived neurotrophic factor and nerve growth factor mRNA in primary cultures of hippocampal neurons and astrocytes.

Brain-derived neurotrophic factor (BDNF) and NGF are both expressed by neurons in the hippocampus. In previous studies, it has been demonstrated that both BDNF and NGF mRNA levels are regulated by neuronal activity. Upregulation is predominantly regulated by the glutamate (NMDA and non-NMDA receptors); downregulation, predominantly by the GABA system (Zafra et al., 1990, 1991). In neuronal cultures of the rat hippocampus, potassium depolarization and kainic acid-mediated increases in BDNF and NGF mRNA were eliminated in a dose-dependent manner by the calcium channel blocker nifedipine. Conversely, calcium ionophores (Bay-K8644 and ionomycin) augmented BDNF and NGF mRNA levels by a calmodulin-mediated mechanism. In view of the fact that many potential modulators (conventional transmitters and neuropeptides) of neuronal and astrocytic BDNF and NGF mRNA synthesis may act via the adenylate cyclase system, we studied the effect of forskolin, an activator of adenylate cyclase. Indeed, forskolin enhanced the effects of calcium ionophores and kainic acid on BDNF and NGF mRNA levels. Cytokines, such as interleukin-1 and transforming growth factor-beta 1, which have previously been shown to increase NGF mRNA markedly in astrocytes, were without effect on neuronal BDNF and NGF mRNA levels. In contrast to neuronal cultures, where the regulation of BDNF and NGF mRNA was generally very similar, the regulation in astrocytes was distinctly different. All the cytokines that produce a marked increase in NGF mRNA were without effect on astrocyte BDNF mRNA levels, which under basic conditions were below the detection limit. However, norepinephrine produced a marked elevation of BDNF mRNA in astrocytes, an effect that was further enhanced by glutamate receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of the nerve growth factor receptor in primary cultures of neural crest cells

Neural crest cells maintained as primary cultures for 4-7 days demonstrated specific binding of radioiodinated nerve growth factor ([125I]NGF); occasionally, significant binding of ([125I]NGF could be detected in 3-day cultures. Parallel cultures processed for indirect immunofluorescence revealed that the NGF receptor-positive neural crest cells represented a subpopulation of the total cells in culture. Cultures aged 4-7 days demonstrated fluorescent cells which were often grouped as aggregates. Some 3-day cultures contained faintly fluorescent cells. One- and two-day cultures were non-fluorescent. Melanocytes did not appear to bind NGF. Preliminary flow cytofluorometric analysis indicated that ca. 28% of neural crest cells in 5-day cultures possessed specific receptors for NGF.