Removal Of Nerve Growth Factor Research Articles

Puromycin induces apoptosis of developing chick sympathetic neurons in a similar manner to NGF-deprivation.

Survival of sympathetic neurons of chick embryo depends on nerve growth factor (NGF), and removal of NGF from culture medium caused apoptosis. We found that puromycin, an analogue of aminoacyl-tRNA, also induced apoptosis in a manner quite similar to that caused by NGF-deprivation. First, transcriptional inhibitors such as actinomycin D and alpha-amanitin effectively prevented both apoptoses, suggesting a requirement of gene expression. Second, the apoptoses were effectively prevented by translational inhibitors acting on peptidyltransfer reaction, such as anisomycin and blasticidin S. On the other hand, emetine, an inhibitor of translocation of peptidyl-tRNA, was not effective. Finally, NGF-deprivation and puromycin-addition affected the same three phosphorylated proteins to undergo dephosphorylation or to be eliminated. Besides, these changes were suppressed by anisomycin and blasticidin, but not by emetine. Based on these findings, we discuss the mechanism to induce apoptosis of chick sympathetic neurons.

Ciliary neurotrophic factor supports p75NGFR-immunoreactive non-cholinergic, but not cholinergic, developing septal neurons in vitro.

Ciliary neurotrophic factor is known to exert both survival and differentiative actions on a number of neuronal populations of the peripheral and central nervous systems. In this study we have compared the trophic effects of ciliary neurotrophic factor and nerve growth factor on developing septal neurons of the rat in vitro. Fetal septal neurons were grown in vitro under glass coverslips in sandwich culture. Septal cultures grown for 14 days in the continual presence of nerve growth factor contain a population of cholinergic neurons that stain intensely for the low-affinity nerve growth factor receptor (p75NGFR), choline acetyltransferase and acetylcholinesterase. Without added nerve growth factor, few neurons stain for these markers. Ciliary neurotrophic factor addition for 14 days from plating in the absence of exogenous nerve growth factor results in the appearance of a population of neurons that stains for p75NGFR. This population is similar in number to that seen in nerve growth factor-treated cultures but is not immunoreactive for choline acetyltransferase and is significantly smaller in mean cross-sectional area. Delayed addition of nerve growth factor to ciliary neurotrophic factor-supported cultures at 14 days for a further seven days fails to induce choline acetyltransferase immunoreactivity in these p75NGFR-positive septal neurons. In cultures grown in the continual presence of nerve growth factor from plating, removal of nerve growth factor and addition of nerve growth factor antibodies at 14 days results in the death of over 80% of the cholinergic neurons after a further four days. Addition of ciliary neurotrophic factor during the period of nerve growth factor withdrawal appears to preserve a p75NGFR-positive, choline acetyltransferase-negative neuronal population. However, seven day re-addition of nerve growth factor to ciliary neurotrophic factor-treated, nerve growth factor-withdrawn cultures fails to induce choline acetyltransferase immunoreactivity in the ciliary neurotrophic factor-supported p75NGFR-positive septal neurons. Simultaneous treatment of cultures with both ciliary neurotrophic factor and nerve growth factor for 14 days from plating approximately doubles the number of p75NGFR-positive neurons relative to cultures treated with either ciliary neurotrophic factor or nerve growth factor alone, but the number of choline acetyltransferase-positive neurons in these cultures is not significantly greater than that found in cultures treated solely with nerve growth factor. These results suggest that ciliary neurotrophic factor does not support the survival and differentiation of developing septal cholinergic neurons in vitro, but can support the development of a p75NGFR-immunoreactive population of non-cholinergic septal neurons.

Long term herpes simplex virus type 1 infection of nerve growth factor-treated PC12 cells

The behaviour of herpes simplex virus type 1 (HSV-1) strain 17 in tissue cultures of PC12 cells treated with nerve growth factor (NGF) was studied. PC12 cells respond to NGF by ceasing to proliferate and extending long neurites. After differentiation with NGF, cultures were infected with HSV-1 and maintained in the presence of the hormone for several weeks. These long-term infected cultures were tested for HSV DNA, transcripts and the ability to produce virus, before and after NGF removal. Before NGF removal, the cultures were characterized by little or no virus production and the presence of HSV-1 DNA in a predominantly endless form. In situ analysis of long-term infected cultures revealed latency-associated transcript expression in only a portion of the cells. However, as shown by an infectious centre assay, virus was present in almost all cells in the population. Moreover, removal of NGF from long-term cultures resulted in the appearance of significantly increased amounts of virus in the media. The degree to which this system resembles HSV latency in vivo is discussed.

Time course of nerve growth factor modulation of ethanol inhibition of Ca 2+ currents in PC12 cells

The effects of 25 mM ethanol on high voltage-activated (HVA) L-type Ca 2+ channels in nerve growth factor (NGF)-treated, dibutyryl cyclic AMP (dbcAMP)-treated and undifferentiated (UND) PC12 cells were examined. Channels in NGF-treated cells became less sensitive to the effects of ethanol. The time course of the NGF-induced reduction in ethanol inhibition occurred in two phases. The first phase of reduced inhibition was observed after 2 h of NGF exposure and was complete after 5 h. The second phase of reduced inhibition developed over a period of 10–96 h and was reversible after removal of NGF. Exposure to dbcAMP for 2 h also caused a transient drop in ethanol inhibition, but the second phase of decreased ethanol sensitivity was absent. Thus, increased levels of cAMP do not appear to be involved in the longer-term effects of NGF on the decrease in ethanol inhibition of Ca 2+ channels, whereas the effects observed at 2 h may involve cAMP.

Nerve growth factor regulates the subcellular localization of the nerve growth factor‐inducible protein PC4 in PC12 cells

The immediate early gene (IEG) PC4, which encodes a protein related to gamma interferon, is activated at the onset of the neuronal differentiation induced by nerve growth factor (NGF) in PC12 cells. With an antibody raised to a bacterial beta gal-PC4 fusion protein, the PC4 protein is detected as an immunoreactive molecular species of 49 kDa, whose synthesis is rapidly induced by NGF in parallel with the induction of its mRNA. Immunofluorescence, electron microscopy and subfractionation studies indicate that the PC4 immunoreactivity is localized in the cytoplasm of PC12 cells, where it is increased transiently by NGF within 3 hr of treatment. In addition, the PC4 immunoreactivity presents an NGF-dependent pattern of intracellular localization. In fact, within 3 hr after addition of NGF, PC4 is also significantly expressed on the inner face of the plasma membrane, to which it is physically associated. After longer NGF treatment, PC4 disappears from the plasma membrane and appears in the nucleus, with reduced cytoplasmic expression. Localization in the nucleus is reversed by removal of NGF and closely parallels changes in the state of differentiation of the cell. The existence within the PC4 protein of a consensus sequence for the addition of myristic acid and of a putative sequence for the nuclear localization suggests possible mechanisms for the NGF-dependent redistribution. For an NGF-inducible IEG product, such growth factor-dependent localization of PC4 is a novel type of regulation in the pathways from the NGF receptor to the adjacent membrane proteins and to the nucleus.

Altered protein binding to the octamer motif appears to be an early event in programmed neuronal cell death.

Electrophoretic mobility-shift assays were used to characterize binding of nuclear proteins to consensus sequences for Sp1, E2F, octamer, and cAMP responsive enhancer element (CRE) during neuronal death in vitro after removal of nerve growth factor (NGF). Molecular events occurring prior to cell death in terminally differentiated PC12 cells could be divided into three phases: (i) within 2 hr of removing NGF, binding to the octamer sequence decreased, (ii) after 5-7 hr an increase in binding to CRE occurred; and (iii) after 14 hr (the point at which 50% of the cells are committed to die) a decrease in binding to the Sp1 sequence occurred. Assays performed with extracts from sympathetic ganglia indicated that changes in binding to CRE and octamer motifs also occurred during the period of developmental cell death in vivo. Double-stranded oligonucleotides were delivered to neurons to act as dominant negative "promoters" unable to couple to transcriptional events but capable of binding and sequestering transcription factors. Double-stranded but not single-stranded octamer oligonucleotides increased cell death of primary cultures of sympathetic neurons. Most of the induced neuronal cell death could be blocked with NGF, which is consistent with oligonucleotides activating an endogenous death program rather than having a nonspecific toxic effect. Other double-stranded oligonucleotides as well as a mutant octamer oligonucleotide had little or no effect on cell death. These data are consistent with the hypothesis that cell death results from a cascade of cellular and molecular events and that an early event in programmed neuronal cell death is a decrease in binding of transcription factor(s) to octamer motif sequences.

Further characterization of scrapie replication in PC12 cells

The rat pheochromocytoma cell line, PC12, undergoes neuron-like morphological, biochemical and electrophysiological differentiation, in the presence of low concentrations of nerve growth factor (NGF). NGF-treated PC12 cells have been shown previously to support 139A scrapie agent replication. In the present report we extended these findings and analysed the cellular conditions necessary for agent replication. Following the infection of differentiated PC12 cells, scrapie replicated to relatively high titres as determined by an incubation period assay. The removal of NGF, which causes the gradual dedifferentiation of PC12 cells, resulted in the inability of scrapie to replicate. The scrapie infectivity detected in PC12 cultures is cell-associated and not released into the medium. Cells in infected cultures did not show any change in morphology when compared to cells in mock-infected cultures. Titration studies of scrapie infectivity in PC12 cells have indicated that up to 4 LD50 units per cell can be obtained although a yield of 1 LD50 per cell was more common. Using an approximate m.o.i. of 1, only differentiated PC12 cells supported 139A scrapie agent replication when compared to two other differentiated, neuronal cell types, indicating that PC12 cells are more susceptible to agent replication. These studies support further the suitability of using differentiated PC12 cells as an in vitro model to study scrapie agent replication.

Nerve growth factor-induced decrease in the calpain activity of PC12 cells

PC12 cells are a nerve growth factor-responsive clone derived from a rat pheochromocytoma. Treatment with nerve growth factor causes the cells to differentiate. One of the hallmarks of this differentiation is the generation of neurites. PC12 cells contain both calpain I and calpain II; about 90% of the total calpain activity is due to calpain II. Treatment of the cells with nerve growth factor causes a time-dependent decrease in calpain activity, more than 50% being lost over a 5-day period. Both the decrease in calpain activity and the growth of neurites are reversible upon the removal of nerve growth factor from the cultures. Agents other than nerve growth factor that cause neurite outgrowth, such as fibroblast growth factor and dibutyryl cyclic AMP, also cause a decrease in calpain activity. Calpain levels, as detected with immunoblotting or immunohistochemistry, show no decrease. Removal of calpastatin, the endogenous inhibitor of the calpains, by phenyl-Sepharose chromatography increases the calpain activity of extracts from both control and nerve growth factor-treated cells and brings the activity in the extracts from treated cells up to the activity in those from controls. Calpastatin-containing fractions from extracts of nerve growth factor-treated cells inhibit more calpain activity than do comparable fractions from control cells. These studies suggest that nerve growth factor causes a decrease in the activity of calpain in morphologically differentiating PC12 cells by causing an increase in the activity of calpastatin.

Neuropeptide expression in cultures of adult sensory neurons: Modulation of substance P and calcitonin gene-related peptide levels by nerve growth factor

In contrast to developing sensory neurons, the survival of adult rat dorsal root ganglion neurons in pure neuronal culture is not dependent on specific neurotrophic factors such as nerve growth factor or brain-derived neurotrophic factor [ Lindsay R.M. (1988) J. Neurosci. 8, 2394–2405]. In the present study we have examined possible modulatory effects of nerve growth factor on the neuropeptide content of sub-populations of adult rat dorsal root ganglion neurons in vitro. During the first 1–2 days in culture the neuropeptides substance P and calcitonin gene-related peptide could be detected by immunofluorescence staining in cultures grown in the presence or absence of nerve growth factor, but at longer times in nerve growth factor-deprived cultures there was loss of immunoreactive staining for both peptides. In the presence of nerve growth factor, however, the percentage of substance P- and calcitonin gene-related peptide-immunoreactive neurons remained relatively constant, for at least 14 days, at levels that were similar to the percentage of such peptide-containing neurons found in sections of adult rat dorsal root ganglia. Quantitation by radioimmunoassay of the levels of substance P and calcitonin gene-related peptide in cultures grown in the presence or absence of nerve growth factor agreed with the qualitative observations obtained by immunofluorescence: 10–15-fold higher levels of substance P and calcitonin gene-related peptide were found in cultures grown with nerve growth factor for 18 days, as compared to nerve growth factor-deprived cultures. In nerve growth factor-treated cultures increased levels of substance P and calcitonin gene-related peptide were observed within 3–6 days in vitro, and further steady increases in the levels of both peptides were found up to 18 days. A low basal level of both peptides could always be detected, even in the presence of an excess of antibodies to nerve growth factor. Up-regulation of the synthesis of substance P and calcitonin gene-related peptide did not depend on nerve growth factor being present at the initiation of the cultures, as elevated levels of both peptides could be induced in cultures even after up to 10 days' prior deprivation of nerve growth factor. Removal of nerve growth factor from the cultures resulted in reduced levels of peptide within 3 days. Taken together these results confirm and extend earlier studies in vivo which have suggested that nerve growth factor may play a dynamic role in the regulation of the levels of substance P in mature sensory neurons. In the present study, it has been established that the effects of nerve growth factor are not related to survival and that substance P is not the only neuropeptide whose level of synthesis is modulated by nerve growth factor.

Intraspinal sprouting after administration of nerve growth factor antibodies to neonatal rats

Previous observations show that neonatal removal of nerve growth factor results in an increased number of fine dorsal root axons in the adult animal. The present study shows that the same treatment results in 49% more axons in the adult tract of Lissauer, which is an area of spinal white matter that contains predominant fine primary afferentaxons. These data are consistent with the idea that postnatal removal of NGF leads to increased numbers of fine primary afferent axons which then pass into the white matter of the spinal cord and there make synaptic contacts. If so, this would be a useful preparation for studies on the mechanisms by which newly formed sensory axons can be induced to enter the spinal cord postnatally.

Developmental changes of nerve growth factor and its mRNA in the rat hippocampus: Comparison with choline acetyltransferase

Previous experiments have demonstrated that in the septo-hippocampal system choline acetyltransferase (ChAT) is induced by nerve growth factor (NGF) (Gnahn et al. (1983) Dev. Brain Res. 9, 45–52) and that hippocampal NGF and mRNA NGF levels are correlated with the density of cholinergic innervation (Korsching et al. (1985) EMBO J. 4, 1389–1393). In the present investigation we have compared the developmental changes of ChAT, NGF, and mRNA NGF levels in this system. During the postnatal development of the hippocampus the time courses of NGF and ChAT were well correlated including the most rapid increase between P12 and P14. This increase in hippocampal NGF was preceded by a corresponding increase in mRNA NGF. The developmental changes in hippocampal NGF levels were also closely reflected by corresponding changes in the septum. This, together with previous observations (Korsching et al., 1985) that the adult septum, in spite of relatively high NGF levels, does not contain measurable quantities of mRNA NGF, suggests that the NGF levels in the septum are determined by the quantity of NGF transported retrogradely from the field of innervation rather than by local synthesis. During the prenatal period hippocampal NGF levels were relatively high, whereas the mRNA NGF was below the level of detection. Since the ingrowth of septal fibers, and with that also the removal of NGF by retrograde transport, begins around birth, the relatively high prenatal NGF levels probably result from an accumulation produced by a small copy number of mRNA NGF prior to the removal of NGF by retrograde axonal transport. It is concluded that the correlation of the developmental changes in NGF and mRNA NGF with the ChAT activity in the hippocampus further supports the concept of a physiological role of NGF in the central nervous system.

Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells.

Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. We show here that within 5 min after its addition, NGF transiently stimulates c-fos proto-oncogene and actin transcription by greater than 100-fold in nonsynchronized PC12 cells. c-myc and ornithine decarboxylase transcription are also transiently activated, but more slowly. The corresponding mRNAs are induced as well. Two weeks' exposure to NGF causes no significant changes in the transcription of these and a variety of other genes analyzed; however, c-fos mRNA levels are increased severalfold under these conditions. Neuronally differentiated PC12 cells retain the capacity for rapid transcriptional responses. Removal of NGF from such cells for several hours followed by its readdition results in rapid induction of c-fos and actin transcription. These NGF-promoted transcriptional changes in PC12 cells are similar to those previously observed in quiescent fibroblasts stimulated by platelet-derived growth factor (Greenberg, M.E., and Ziff, E.B. (1984) Nature 311, 433-438). This rapid transcriptional activation in PC12 cells could be necessary for neuronal differentiation, but is apparently not sufficient since diverse agents without differentiating activity such as epidermal growth factor, insulin, dibutyryl cAMP, phorbol ester, and elevated K+ were also found to induce transcription. These results suggest that c-fos, c-myc, and actin induction may be general nuclear responses to growth or differentiation factors in a variety of different cell types.

Nerve growth factor supply for sensory neurons: Site of origin and competition with the sympathetic nervous system

Nerve growth factor (NGF) levels in the sensory nervous system were measured by a highly sensitive two-site enzyme immunoassay for NGF. Dorsal root ganglia and the adjacent spinal nerves contained 2.8 +/- 0.3 and 1.7 +/- 0.4 ng NGF/g wet wt., respectively, whereas no NGF was detectable in dorsal roots and spinal cord (less than 0.05 ng NGF/g wet wt.). It is concluded that sensory neurons are supplied with NGF exclusively from their peripheral and not from their central field of projection. Two days after treatment with 6-hydroxydopamine, which destroys sympathetic nerve terminals and thereby prevents the removal of NGF by sympathetic neurons, the NGF content of dorsal root ganglia and trigeminal ganglia increased to 285% and 161% of control, respectively. This indicates that in peripheral target tissues sensory and sympathetic neurons compete for NGF.

Regulation of growth cone morphology by nerve growth factor: A comparative study by scanning electron microscopy

The object of this study was to document and analyze local regulation by nerve growth factor (NGF) of neuronal growth cone properties and to explore the possible diversity of this effect in various NGF-responsive preparations. In particular, scanning electron microscopy was used to characterize the morphology of neuronal growth cones in cultures of dissociated chick embryo dorsal root ganglia (DRG) under conditions of continuous NGF exposure, withdrawal of NGF for 5-6 hr, and restoration of NGF for various times. Comparison was made with similarly manipulated cultures of dissociated newborn rat sympathetic ganglia and neurite-bearing PC12 pheochromocytoma cells. The growth cones of most of the continuously NGF-treated DRG neurons (cultured on poly-L-lysine or collagen-coated glass coverslips) had relatively compact central flattened areas and numerous prominent filopodia. Withdrawal of NGF resulted in a marked spreading of the central growth cone area so that the average maximum width of this structure increased by about threefold as compared to nondeprived cultures. The mean number and lengths of filopodia were unaffected. Restoration of NGF brought about, over a time course of tens of minutes, a return of the original type of growth cone morphology. Rather different responses were observed for the sympathetic neuron and PC12 cultures. Here, surface ruffles, only rarely seen in the chick cultures, were a major feature of the growth cones, whereas filopodia, though present, were less prominent. Removal of NGF led to loss of ruffles and to rounding up of the growth cones; NGF readdition elicited a rapid (less than 30 sec) reinitiation of ruffling and a more gradual (over tens of minutes) respreading of growth cones. These findings illustrate not only that NGF can regulate growth cone properties, but also that there is a diversity as to how this is manifested. Possible mechanisms and biological roles for this regulation are discussed.

The level of nerve growth factor (NGF) as a function of innervation. A correlation radio-immunoassay and bioassay study of the rat iris.

A two-site radio-immunoassay for beta NGF demonstrated 5-10 pg of NGF in the normal, adult rat iris. Ciliarectomy or sympathectomy did not significantly alter the amount of NGF after 10 days. However, denervation including all sensory axons (stereotactic lesion distal to the trigeminal ganglion) increased the level to about 100 pg of NGF. Total denervation resulting from homologous transplantation of the iris gave a similar increase after only 2 days. Fibre outgrowth responses evoked by corresponding iris explants in an NGF bioassay supported the results and suggested in addition that sympathetic denervation may cause a moderate transient increase in NGF after 3 days. It seems that sensory nerves in particular influence the level of NGF in a terminal field, either by a high capacity for uptake and removal of NGF or by exerting a negative feed-back on the production or processing of this growth factor.

Stimulation by nerve growth factor of specific protein synthesis in rat PC12 pheochromocytoma cells

PC12 cells are a clonal line derived from a rat adrenal pheochromocytoma. When treated with ngm/ml levels of nerve growth factor PC12 cells stop dividing, extend long neurities and become electrically excitable. We have used polyacrylamide gradient gel electrophoresis in the presence of sodium dodecylsulphate to study the effects of nerve growth factor treatment on PC12 proteins. Treatment with nerve growth factor has very little effect on the relative abundances of major PC12 proteins. One consistent effect of the factor is induction of a polypeptide (designated p80) of apparent molecular weight 80,000. Induction of p80 is apparent within 1 day after addition of nerve growth factor and thus precedes neurite outgrowth; maximal induction is reached by 2–3 days of nerve growth factor treatment. Induction of p80 appears to be due to increased polypeptide synthesis rather than to increased stability of pre-existing molecules. Induction is rapidly reversible upon removal of nerve growth factor from cells pretreated with the factor. Induction and maintenance of p80 synthesis by nerve growth factor are selectively sensitive to camptothecin, an inhibitor of ribonucleic acid synthesis. Induction of p80 occurs normally in cells treated with nerve growth factor when process outgrowth is blocked by anti-microtubule agents or by growth in suspension. Induction is also normal when cells are treated with nerve growth factor in the absence of serum. The results suggest that transcription-dependent induction of specific protein synthesis is an early effect of nerve growth factor on PC12 cells and may play a role in bringing about nerve growth factor-induced changes in cell shape, division and physiological properties.

Local control of neurite development by nerve growth factor

A three-chamber culture system was devised in which neurites growing from small clusters of somas of sympathetic neurons penetrated a virtually fluid-impermeable barrier; thus the local fluid environment of the distal portions of the neurites could be controlled independently of the local fluid environment of the somas and proximal portions of the neurites. Neurites regularly penetrated the barriers if a high concentration of nerve growth factor was present on both sides, but never penetrated into chambers to which no nerve growth factor had been added. After neurites crossed the barrier, local removal of nerve growth factor from the distal portions of the neurites caused the growth of these portions to stop, and they eventually appeared to degenerate even though nerve growth factor was continuously present in the chamber that contained their somas and proximal portions. In contrast, local nerve growth factor was not required at the somas and proximal portions of the neurites; many neurons survived its withdrawal provided their somas were associated with neurite bundles that crossed into a chamber containing nerve growth factor. These results show that the growth, and probably the survival, of neurites depends upon nerve growth factor in their local environment, regardless of the nerve growth factor concentrations to which other portions of the neuron are exposed. This is entirely consistent with the notion that nerve growth factor released by sympathetic target tissues promotes the establishment and maintenance of appropriate neuron-target connections during development.

Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.

A single cell clonal line which responds reversibly to nerve growth factor (NGF) has been established from a transplantable rat adrenal pheochromocytoma. This line, designated PC12, has a homogeneous and near-diploid chromosome number of 40. By 1 week's exposure to NGF, PC12 cells cease to multiply and begin to extend branching varicose processes similar to those produced by sympathetic neurons in primary cell culture. By several weeks of exposure to NGF, the PC12 processes reach 500-1000 mum in length. Removal of NGF is followed by degeneration of processes within 24 hr and by resumption of cell multiplication within 72 hr. PC12 cells grown with or without NGF contain dense core chromaffin-like granules up to 350 nm in diameter. The NGF-treated cells also contain small vesicles which accumulate in process varicosities and endings. PC12 cells synthesize and store the catecholamine neurotransmitters dopamine and norepinephrine. The levels (per mg of protein) of catecholamines and of the their synthetic enzymes in PC12 cells are comparable to or higher than those found in rat adrenals. NGF-treatment of PC12 cells results in no change in the levels of catecholamines or of their synthetic enzymes when expressed on a per cell basis, but does result in a 4- to 6-fold decrease in levels when expressed on a per mg of protein basis. PC12 cells do not synthesize epinephrine and cannot be induced to do so by treatment with dexamethasone. The PC12 cell line should be a useful model system for neurobiological and neurochemical studies.