Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection

The intact sciatic nerve contains levels of nerve growth factor (NGF) that are comparable to those of densely innervated peripheral target tissues of NGF-responsive (sympathetic and sensory) neurons. There, the high NGF levels are reflected by correspondingly high mRNANGF levels. In the intact sciatic nerve, mRNANGF levels were very low, thus indicating that the contribution of locally synthesized NGF by nonneuronal cells is small. However, after transection an increase of up to 15-fold in mRNANGF was measured in 4-mm segments collected both proximally and distally to the transection site. Distally to the transection site, augmented mRNANGF levels occurred in all three 4-mm segments from 6 h to 2 wk after transection, the longest time period investigated. The augmented local NGF synthesis after transection was accompanied by a reexpression of NGF receptors by Schwann cells (NGF receptors normally disappear shortly after birth). Proximal to the transection site, the augmented NGF synthesis was restricted to the very end of the nerve stump that acts as a "substitute target organ" for the regenerating NGF-responsive nerve fibers. While the mRNANGF levels in the nerve stump correspond to those of a densely innervated peripheral organ, the volume is too small to fully replace the lacking supply from the periphery. This is reflected by the fact that in the more proximal part of the transected sciatic nerve, where mRNANGF remained unchanged, the NGF levels reached only 40% of control values. In situ hybridization experiments demonstrated that after transection all nonneuronal cells express mRNANGF and not only those ensheathing the nerve fibers of NGF-responsive neurons.     

Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells.

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.     

Sympathetic axons surround nerve growth factor-immunoreactive trigeminal neurons: Observations in mice overexpressing nerve growth factor

It has been postulated that the aberrant projection of sympathetic axons to individual primary sensory neurons may provide the morphological basis for pain-related behaviors in rat models of chronic pain syndrome. Since nerve growth factor (NGF) can elicit the collateral sprouting of noradrenergic sympathetic terminals, it might be predicted that NGF plays a role in mediating the sprouting of sympathetic axons into sensory ganglia. Using a line of transgenic mice overexpressing NGF among glial cells, it was first found that trigeminal ganglia from adult transgenic mice possessed significantly higher levels of NGF protein in comparison to age-matched wild-type mice; as well, detectable levels of NGF mRNA transgene expression were present in both the ganglia and brain stem. Within the trigeminal ganglia, a small proportion of the sensory neuronal population stained immunohistochemically for NGF; a higher percentage of NGF-positive neurons was evident in transgenic mice. New sympathetic axons extended into the trigeminal ganglia of transgenic mice only and formed perineuronal plexuses surrounding only those neurons immunostained for NGF. In addition, such plexuses were accompanied by glial processes from nonmyelinating Schwann cells. From these data, we propose that accumulation of glial-derived NGF by adult sensory neurons and its putative release into the ganglionic environment induce the directional growth of sympathetic axons to the source of NGF, namely, the cell bodies of primary sensory neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 347–360, 1998     

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 mRNANGF 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 mRNANGF 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 mRNANGF. 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 mRNANGF, 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 mRNANGF 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 mRNANGF prior to the removal of NGF by retrograde axonal transport. It is concluded that the correlation of the developmental changes in NGF and mRNANGF with the ChAT activity in the hippocampus further supports the concept of a physiological role of NGF in the central nervous system.     

CHANGES OF ENZYME PATTERN IN THE SYMPATHETIC NERVOUS SYSTEM OF ADULT MICE AFTER SUBMAXILLARY GLAND REMOVAL; RESPONSE TO EXOGENOUS NERVE GROWTH FACTOR

—Removal of the submaxillary glands, the apparent site of NGF synthesis in adult mice, caused a decrease in the activity of all the enzymes involved in the biosynthesis of noradrenaline in the peripheral sympathetic nervous system. Thus, tyrosine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) DOPA decarboxylase (EC 4.1.1.28.) and dopamine β-hydroxylase (EC 1.14.17.1.) showed reduced activity 10 days after removal of the submaxillary glands in both superior cervical and stellate ganglia. This decrease in enzyme activity persisted up to 100 days after surgery. The fourth enzyme studied, choline acetyl-transferase (EC 2.3.1.6.) which is exclusively localized within the presynaptic cholinergic terminals of the ganglia was not affected by sialectomy. A dose of 50 μg NGF/animal/day given over 4 days was only able to restore the enzyme activity to control levels in the superior cervical ganglia of sialectomized mice whereas in stellate ganglia the enzyme activities rose above control levels to a similar extent in sialectomized and non-sialectomized animals. These results provide biochemical evidence that NGF may play a role not only during the growth and normal development of the peripheral sympathetic nervous system but also in the maintenance of its functional integrity in the adult animal.     

Studies on the regulation of beta-nerve growth factor gene expression in the rat iris: the level of mRNA-encoding nerve growth factor is increased in irises placed in explant cultures in vitro, but not in irises deprived of sensory or sympathetic innervation in vivo

Beta-nerve growth factor (NGF) is a protein necessary for the survival and maintenance of sympathetic and sensory neurons that appears to be produced by the target tissues of these neurons in vivo. Both denervation and the culture of explants of one model target, the rat iris, leads to an increase in the NGF content, suggesting that innervating neurons may regulate a step in synthesis or turnover of NGF. To determine whether there is a change in synthesis controlled at the mRNA level, the rat iris has been assayed for its content of NGF mRNA after surgical and chemical denervation and after explant into culture. Using a sensitive blot hybridization assay, a large, rapid increase in the content of NGF mRNA was observed upon explant of the rat iris. The increase was readily detectable within 1 h, reached a maximum increase of 10- to 20-fold by 6 to 12 h, and was still evident after 3 d in culture. The distribution of NGF mRNA in different areas of the iris does not change during this time. This rapid increase in NGF mRNA is also seen in the fully innervated iris in vivo after trauma to the anterior chamber. In contrast, denervation to varying degrees in situ had no effect on NGF mRNA levels. Neither removal of sympathetic innervation by surgical or chemical methods nor combined surgical removal of sympathetic and sensory innervation detectably altered NGF mRNA content. Thus, denervation of the rat iris in situ does not cause the observed accumulation of NGF by increasing the level of NGF mRNA, and the increase in NGF content must be due to other factors.     

Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.

The levels of nerve growth factor (NGF) and its mRNA in the rat central nervous system were determined by two-site enzyme immunoassay and quantitative Northern blots, respectively. Relatively high NGF levels (0.4-1.4 ng NGF/g wet weight) were found both in the regions innervated by the magnocellular cholinergic neurons of the basal forebrain (hippocampus, olfactory bulb, neocortex) and in the regions containing the cell bodies of these neurons (septum, nucleus of the diagonal band of Broca, nucleus basalis of Meynert). Comparatively low, but significant NGF levels (0.07-0.21 ng NGF/g wet weight) were found in various other brain regions. mRNANGF was found in the hippocampus and cortex but not in the septum. This suggests that magnocellular cholinergic neurons of the basal forebrain are supplied with NGF via retrograde axonal transport from their fields of innervation. These results, taken together with those of previous studies showing that these neurons are responsive to NGF, support the concept that NGF acts as trophic factor for magnocellular cholinergic neurons.     

Comparison between the time course of changes in nerve growth factor protein levels and those of its messenger RNA in the cultured rat iris

In previous experiments, it has been demonstrated that, in rat irides in culture, a rapid increase in nerve growth factor (NGF) levels occurred (see Barth, E.-M., Korsching, S., and Thoenen, H. (1984) J. Cell Biol. 99, 839-843). We have now determined the levels of mRNANGF in rat irides as a function of time in culture as well. After an initial lag period of 2 h, mRNANGF levels were transiently increased, so that after 12 h, they had increased 35-fold with respect to zero time. In contrast, poly(A)+ RNA levels dropped to 55% of the zero time values within 5 h, recovered to 85% after 24 h, and remained constant until the end of the observation period. Total ribosomal RNA was found to remain constant, indicating that there was no nonspecific decline of overall metabolic function. Actinomycin D prevented the increase in mRNANGF without reducing the basic mRNANGF levels over a 5-h time period, indicating that the enhanced synthesis of NGF in the rat iris in culture is primarily mediated by an augmented production of mRNANGF. The increases of mRNANGF, cellular NGF, and NGF released into the medium were found to be strictly sequential. Monensin selectively abolished the increased production of mature NGF (see Barth et al.) but not of mRNANGF, suggesting that the processing of NGF precursor is prevented.     

Effects of Nerve Growth Factor on Glutathione Peroxidase and Catalase in PC 12 Cells

Abstract: Nerve growth factor (NGF) is a member of the neuro- trophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione per- oxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC 12 cells in a time- and dose-dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.     

Structure and expression of the chicken beta nerve growth factor gene.

The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult.     

The production and storage of Nerve Growth Factor in vivo by tissues of the mouse,rat, guinea pig,hamster, and gerbil

The Nerve Growth Factor (NGF) content in vivo of tissues from the mouse and rat at various stages of development from 3 days embryonic gestation to the attainment of full maturity has been determined using the standard biological assay. A less extensive survey has also been made of tissues from the guinea pig, hamster, and gerbil. With the exception of the well-documented high levels of NGF in the mouse submaxillary glands, none of the organs examined contained detectable NGF. These results, which are consistent with those previously reported using the biological assay, stand in contrast to the high levels of NGF detected in virtually all tissues by some published radioimmunoassays. It is likely that the discrepancies are due to the use in the radioimmunoassays of antisera containing antibodies to proteins other than NGF, and to the inability of one-site radioimmunoassays to distinguish between the presence of NGF and that of agents capable of binding NGF. The apparent lack of widespread NGF production in vivo contrasts with the ability of many tissues to synthesize the protein in vitro. This may imply that physiologically significant levels of NGF are below the limits of sensitivity of the assay systems presently available, that NGF synthesis in vivo occurs only during a very restricted period of development, or that the presence of a normal innervation pattern influences NGF production.     

Nerve growth factor synthesis in cultured rat iris: modulation by endogenous transmitter substances

Organ cultures of rat iris show a characteristic change in the levels of both nerve growth factor (NGF) and its mRNA: a rapid but transient initial increase is followed by a smaller but persistently elevated NGF synthesis. This time course may be influenced by release of a factor(s) from degenerating nerve terminals and/or by the lack of some factor(s) repressing NGF synthesis in vivo. We therefore analyzed the influence of biogenic amine transmitter substances and putative neuropeptides on this elevation of NGF synthesis in cultured iris. The marked increase of NGF synthesis seen initially in culture was not completely mimicked by any of the substances tested. A specific increase in NGF production up to 150% of control was observed only with cGMP. We also obtained some evidence that reaction to trauma following the culture procedure could enhance NGF production: cutting of irides into small pieces increased NGF production in culture up to 250% of control and, vice versa, treatment with 1 microM dexamethasone decreased NGF production to about 60% of control. However, the sympathetic neurotransmitter norepinephrine (NE) decreased both NGF and its mRNA levels specifically in a dose-dependent manner (0.01-1 mM) to a minimum of about 25% of control. In situ hybridization with mRNA(NGF)-specific probes showed that in cultures of dissociated iris cells all cells were capable of expressing mRNA(NGF), but that 0.1 mM NE preferentially decreased expression of mRNA(NGF) in smooth muscle cells. Thus, our results indicate that the sympathetic transmitter NE is capable of downregulating NGF synthesis in the target cells of sympathetic neurons.     

Denervation, but Not Decentralization, Reduces Nerve Growth Factor Content of the Mesenteric Artery

Abstract: In the present study we applied an improved nerve growth factor (NGF) extraction method to examine the effects of denervation and sympathetic decentralization on NGF levels in vascular tissue. Adult male Wistar Kyoto rats underwent mesenteric arterial denervation or splanchnic nerve transection. Four days after operation, animals were killed, and the mesenteric artery and coeliac-superior mesenteric ganglia were removed. The arterial adventitia was stripped from the media to measure NGF levels in nerve and smooth muscle separately. A high concentration of NGF was detected in the normal artery, 90% of which was in the adventitial layer. Surgical denervation significantly reduced the NGF levels in the artery and ganglia by 78 and 71%, respectively. However, within the artery the level of NGF was reduced in the adventitia but not in the media. Thus, the large reduction of NGF content resulted from the loss of nerve plexus from the artery. In contrast, decentralization did not alter the NGF content in the artery, in either the adventitia or media. Our results are in marked contrast to previous studies reporting elevated levels of NGF following denervation. This discrepancy is explained by the ability of our new procedure to extract much greater amounts of NGF from the tissue.     

Retrograde Transport of Endogenous Nerve Growth Factor in Superior Cervical Ganglion of Adult Rats

The concentration of naturally synthesized nerve growth factor (NGF) was measured in various tissues of adult rats, using a highly sensitive two-site enzyme immunoassay. The highest concentration was found in the superior cervical sympathetic ganglion (SCG). Transection of the postganglionic external carotid nerve (ECN) reduced the ganglionic level of NGF more than did section of the internal carotid nerve (ICN). When both the preganglionic nerve and the ECN were cut, the ganglionic NGF level decreased even more. On the other hand, when the preganglionic nerve and the ICN were both sectioned, leaving the ECN intact, endogenous NGF content in the SCG was significantly enhanced 3-9 h after operation. Bilateral extirpation of submaxillary gland produced a rapid decrease in ganglionic NGF 3-6 h after operation, and even unilateral removal of one salivary gland caused a decrease in both ganglia, which was however much greater in the ipsi- than in the contralateral ganglion. Removal of the eyeballs caused a much smaller reduction in ganglionic NGF than did removal of the glands. These results suggest that the endogenous NGF that accumulates in the SCG is mostly synthesized in the submaxillary gland rather than in the iris, and that it is transported to the SCG, mostly via the ipsilateral ECN.     

Detection of nerve growth factor mRNA in the developing chicken embryo

Nerve growth factor (beta NGF) is a protein supporting sympathetic and sensory innervation in the peripheral tissues as well as cholinergic innervation in the brain. A DNA probe derived from a genomic clone coding for chicken NGF was used to study NGF mRNA levels during development. NGF mRNA was detected in the chicken embryo as early as day 3.5 of incubation. The level of NGF mRNA in total embryo increased four-fold until day 8, remained high until day 12, and subsequently decreased. No corresponding peak in NGF mRNA expression was found in heart and brain measured separately. Instead these organs showed increased NGF mRNA levels after hatching. The highest levels of NGF mRNA in the day-8 embryo were found in skin and eye (in particular cornea, but also iris, sclera-choroid and neural retina) suggesting a correlation between sensory innervation and this early peak of NGF expression.     

Outgrowth of sympathetic adrenergic neurons in mice treated with a nerve-growth factor (NGF)

Summary Various tissues from mice treated with a nerve-growth factor (NGF) were studied with the histochemical technique ofFalck andHillarp, which visualizes the adrenergic transmitter in the sympathetic postganglionic neurons. Growth stimulation was detectable in all parts of the sympathetic adrenergic neurons. An increased density of the adrenergic ground plexus was observed in e.g. the iris, submaxillary and parotid glands, blood vessels and intramural ganglionic plexuses of the intestinal tract. Normally non-innervated tissues were also found to contain a considerable number of adrenergic terminals. Of special interest is the striking increase in number of adrenergic terminals in various types of autonomic ganglia, in all probability with an inhibitory effect on ganglionic transmission.This investigation was supported by research grants from the Swedish Medical Research Council (B67-12x-714-02), Magnus Bergwalls stiftelse and Stiftelsen Therese och Johan Anderssons Minne. The skillful technical assistance of MissBarbro Riese is gratefully acknowledged.     

The Distribution of Nerve Growth Factor in the Male Sex Organs of Mammals

Abstract: The Nerve Growth Factor (NGF) content of male sex organs of the mouse, rat, guinea pig, hamster, rabbit, human, and bull has been investigated using both a biological assay and a two-site radioimmunoassay. The prostate glands of the rabbit and bull have been found to contain moderate levels of NGF, these being lower than the concentrations found in the guinea pig prostate and mouse submaxillary glands. The sex organs investigated of the mouse, rat, hamster, and human contained no detectable NGF activity. Genital organs, other than the prostate glands, of the guinea pig and rabbit were also devoid of NGF. The NGFs from the rabbit and bull are immunologically related to those found in the submaxillary glands of the mouse and the prostate glands of the guinea pig, but immunodiffusion and radioimmunoassay experiments show that there are also clear differences between the NGFs. The use of a two-site radioimmunoassay, based on purified antibodies against mouse submaxillary gland NGF, for the determination of NGF levels in species other than the mouse, is described. It is essential during such applications to compensate for the fact that the NGFs from different species are sufficiently distinct that only part of the antibody population (raised against mouse NGF) is capable of recognizing NGF from species other than the mouse. The results of radioimmunoassay and biological assay determinations are in reasonable agreement, if corrections for this feature are made.     

p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors.

The relative levels of mRNA specific for the mouse p53 cellular tumor antigen were determined in various normal adult tissues, embryos, and tumors. All tumors studied contained concentrations of p53 mRNA well above those present in most normal tissues. Normal spleen, however, had p53 mRNA levels comparable to those found in some tumors, despite the fact that they contained barely detectable p53 protein. This apparent discrepancy was found to be due to the extremely rapid turnover rate of p53 in the spleen (half-life, approximately equal to 6 min). In developing fetuses, a marked reduction of p53 mRNA levels was manifest from day 11 onwards, whereas the levels during organogenesis (days 9 to 11) were comparable to those found in undifferentiated embryonic stem cells and in some tumors.     

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.