Reduced age-related plasticity of neurotrophin receptor expression in selected sympathetic neurons of the rat

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.     

Level of p75 receptor expression in sensory ganglia is modulated by NGF level in the target tissue

Neurotrophins play an essential role in sensory development by providing trophic support to neurons that innervate peripheral targets. Nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, and brain-derived neurotrophin exert their survival effect by binding to two transmembrane receptor types: trk receptors, which exhibit binding specificity, and the p75^NTR receptor, which binds all neurotrophins. To determine how target-derived neurotrophins affect sensory neuron development and function, we used transgenic mice that overexpress NGF in the skin to examine the impact of NGF overexpression on receptor expression. Previous studies of trk expression in trigeminal ganglia of adult NGF transgenics showed that the percentage of trkA neurons doubled and their number increased fivefold. The present study focused on the p75 receptor and shows that the percentage of neurons expressing p75^NTR also increase in NGF ganglia, but only by 10%. This increase did not encompass the small, BS-IB-4 isolectin-positive cells as they remained p75 negative in transgenic ganglia. Interestingly, levels of trkA protein were not increased on a per-cell level, whereas levels of p75^NTR increased nearly threefold. These results show that in sensory systems, target-derived NGF modulates the level of p75^NTR receptor expression, and in so doing, may act to regulate the formation of functional receptor complexes and subsequent trophic action. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 258–270, 1998     

Ectopic trkA expression mediates a NGF survival response in NGF- independent sensory neurons but not in parasympathetic neurons

We have investigated the role of trkA, the tyrosine kinase NGF receptor, in mediating the survival response of embryonic neurons to NGF. Embryonic trigeminal mesencephalic (TMN) neurons, which normally survive in the presence of brain-derived neurotrophic factor (BDNF) but not NGF, become NGF-responsive when microinjected with an expression vector containing trkA cDNA. In contrast, microinjection of ciliary neurotrophic factor (CNTF)-dependent embryonic ciliary neurons with the same construct does not result in the acquisition of NGF responsiveness by these neurons despite de novo expression of trkA mRNA and protein. The failure of trkA to result in an NGF-promoted survival response in ciliary neurons is not due to absence of the low-affinity NGF receptor, p75, in these neurons. Quantitative RT/PCR and immunocytochemistry showed that TMN and ciliary neurons both express p75 mRNA and protein. These findings not only provide the first direct experimental demonstration of trkA mediating a physiological response in an appropriate cell type, namely NGF-promoted survival of embryonic neurons, but indicate that not all neurons are able to respond to a trkA-mediated signal transduction event.     

Subpopulations of rat B2(+) neuroblasts exhibit differential neurotrophin responsiveness during sympathetic development

Sympathetic neurons comprise a population of postmitotic, tyrosine hydroxylase expressing cells whose survival is dependent upon nerve growth factor (NGF) both in vivo and in vitro. However, during development precursors to rat sympathetic neurons in the thoracolumbar region are not responsive to NGF because they lack the signal transducing NGF receptor, trkA. We have previously shown that acquisition of trkA expression is sufficient to confer a functional response to NGF. Here we describe four subpopulations of thoracolumbar sympathetic neuroblasts which are mitotically active and unresponsive to NGF at E13.5 of rat gestation, but differ based upon their neurotrophic responsiveness in vitro. The survival in culture of the largest sympathetic subpopulation is mediated by neurotrophin-3 (NT-3) or glial-derived neurotrophic factor (GDNF), whereas the cell survival of two smaller subpopulations of neuroblasts are mediated by either solely GDNF or solely NT-3. Finally, we identify a subpopulation of sympathetic neuroblasts in the thoracolumbar region whose survival, exit from the cell cycle, induction of trkA expression, and consequent acquisition of NGF responsiveness in culture appear to be neurotrophin independent and cell autonomous. These subpopulations reflect the diversity of neurotrophic actions that occur in the proper development of sympathetic neurons.     

NGF utilizes c-Ret via a novel GFL-independent, inter-RTK signaling mechanism to maintain the trophic status of mature sympathetic neurons.

During postnatal development, sympathetic neurons lose their dependence upon NGF for survival but continue to require NGF for soma and process growth and for development of a mature neurotransmitter phenotype. Although c-Ret is expressed in sympathetic neurons during this period, its function in these transitional processes is unclear. The level of Ret phosphorylation markedly increased with postnatal age in SCG neurons in vitro and in vivo. Postnatal Ret phosphorylation did not require either GFLs or GFR(alpha) coreceptors. Instead, NGF promoted age-dependent Ret phosphorylation with delayed kinetics both in vitro and in vivo. Functionally, maximal NGF-dependent trophism of mature sympathetic neurons required Ret, but not GFR(alpha) coreceptors. Therefore, NGF promotes phosphorylation of the heterologous RTK Ret resulting in augmented growth, metabolism, and gene expression.     

Regulation of nerve growth factor mRNA levels in developing rat heart ventricle is not altered by sympathectomy

The survival of sympathetic and sensory neurons is known to be controlled by nerve growth factor (NGF) supplied by the targets of innervation, yet little is known about how target NGF synthesis is regulated. We have investigated the pattern of NGF mRNA expression in developing rat heart ventricle using a sensitive RNA blotting procedure. We find that the concentration of NGF mRNA increases steadily from Embryonic Day 17 to peak levels at 10-14 days postnatal and then declines about twofold and stabilizes at the level found in adults. The rise in NGF mRNA concentration correlates with the arrival and differentiation of sympathetic nerve terminals in the heart and the cessation of sympathetic cell death. To assess the role of innervating sympathetic neurons in regulating NGF mRNA expression, neonatal rats were sympathectomized by treatment with 6-hydroxydopamine and heart ventricles were assayed for NGF message. Although this treatment reduced ventricle norepinephrine content by 82%, no significant change in NGF mRNA concentration was observed. These results suggest that the developmental program of NGF mRNA production in the heart is not influenced by innervating sympathetic neurons.     

NGF gene expression and secretion in white adipose tissue: regulation in 3T3-L1 adipocytes by hormones and inflammatory cytokines

The sympathetic nervous system plays a central role in lipolysis and the production of leptin in white adipose tissue (WAT). In this study, we have examined whether nerve growth factor (NGF), a target-derived neurotropin that is a key signal in the development and survival of sympathetic neurons, is expressed and secreted by white adipocytes. NGF mRNA was detected by RT-PCR in the major WAT depots of mice (epididymal, perirenal, omental, mesenteric, subcutaneous) and in human fat (subcutaneous, omental). In mouse WAT, NGF expression was observed in mature adipocytes and in stromal vascular cells. NGF expression was also evident in 3T3-L1 cells before and after differentiation into adipocytes. NGF protein, measured by ELISA, was secreted from 3T3-L1 cells, release being higher before differentiation. Addition of the sympathetic agonists norepinephrine, isoprenaline, or BRL-37344 (beta(3)-agonist) led to falls in NGF gene expression and secretion by 3T3-L1 adipocytes, as did IL-6 and the PPARgamma agonist rosiglitazone. A substantial decrease in NGF expression and secretion occurred with dexamethasone. In contrast, LPS increased NGF mRNA levels and NGF secretion. A major increase in NGF mRNA level (9-fold) and NGF secretion (相似文献   

Developmental changes of nerve growth factor levels in sympathetic ganglia and their target organs

The predominant source of nerve growth factor (NGF) used by mature sympathetic neurons originates in their target organs (Heumann, R., Korsching, S., Scott, J., and Thoenen, H. (1984), EMBO J. 3, 3183-3189; Korsching, S., and Thoenen, H. (1985), J. Neurosci. 5, 1058-1061). We have determined the NGF content of two sympathetically innervated mouse organs, submandibular gland and heart ventricle, and of sympathetic ganglia from mouse and rat between embryonic Day 12 (E12) and adulthood. NGF levels were measured by a two-site enzyme immunassay (Korsching, S., and Thoenen, H. (1983), Proc. Natl. Acad. Sci. USA 80, 3513-3516). In heart ventricle and submandibular gland, NGF first became detectable around the time of initial innervation by sympathetic neurons (E12 and E13, respectively) and increased respectively 14- and 7-fold in the following 2 days, to reach adult levels already at E14 for heart ventricle (1.4 +/- 0.2 ng NGF/g wet wt). NGF in the superior cervical ganglion (SCG) was first detected at the same time as in its target organ, the submandibular gland. NGF content in the SCG then increased 6-fold during the next 2 days and continued to increase until the end of the third postnatal week, when adult levels were reached. Although the levels of NGF in the adult mouse submandibular gland are sexually dimorphic and six orders of magnitude higher than those in other sympathetic target organs, no sex difference in the NGF content was found in either developing submandibular gland or SCG until the end of the third postnatal week. Moreover, the steep NGF increase observed in the male submandibular gland after postnatal Day 18 (250-fold within the following 3 days and up to the 55,000-fold in the next 7 days) was not reflected in a corresponding increase in the NGF content of the male SCG. These data indicate that, in accordance with earlier findings (see Levi-Montalcini, R., and Angeletti, P. U. (1968), Physiol. Rev. 48, 534-569), SCG neurons do not have access to the large amounts of NGF synthesized during and after adolescence in the mouse submandibular gland. Our results support the concept that initial fiber outgrowth of sympathetic neurons is neither dependent on NGF nor mediated by it. The time course of NGF levels in the SCG is consistent with the concept that sympathetic neurons are provided with NGF by means of retrograde axonal transport from the innervated organs already early in development.     

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.     

Female steroid hormones modulate receptors for nerve growth factor in rat dorsal root ganglia

Calcitonin gene-related peptide (CGRP) is a vasodilatory peptide, and it is primarily synthesized in dorsal root ganglia (DRG). Plasma CGRP levels increase during pregnancy and with steroid hormones, and nerve growth factor (NGF) stimulates calcitonin/CGRP promoter and CGRP synthesis in DRG. We previously showed that CGRP levels in DRG were stimulated with steroid hormone treatments in vivo but not in vitro. Thus, the stimulation of CGRP by these hormones may be indirect through the upregulation of NGF effects. We hypothesized that the female sex steroid hormones upregulate NGF receptors, trkA and p75(NTR), in DRG. We examined the effects of 17 beta-estradiol (E(2)) and progesterone (P(4)) on NGF receptors in DRG obtained from ovariectomized (ovx) rats. Groups of 4 ovx rats were injected s.c. with 5 microg E(2), 4 mg P(4), or 5 microg E(2) + 4 mg P(4) in 0.2 ml sesame oil or injected with oil only and were killed at 6, 24, and 48 h. In addition, ovx rats were also injected s.c. with varying doses (0.2, 1.0, 5.0, 25 microg) of E(2) (0.5, 1.5, 4, 10 mg) P(4), and (5 microg) E(2) + (0.5, 1.5, 4.0, 10 mg) P(4) in 0.2 ml sesame oil, or vehicle, and killed at 6 (for E(2)) or 24 (for P(4) and E(2) + P(4)) h. Furthermore, groups of ovx rats were also killed at 12 and 24 h; 3 and 7 days; 2, 4, and 6 wk after ovariectomy. The DRGs were collected from all groups and then processed for Western immunoblotting to examine both trkA and p75(NTR) levels. Estradiol increased trkA at 6 h but not p75(NTR). Progesterone caused upregulation of trkA and p75(NTR) at 6 and 24 h. 17 beta-Estradiol + P(4) increased trkA at 6 and 24 h and p75(NTR) at all time points examined. One microgram of E(2) increased trkA but did not affect p75(NTR) levels. Progesterone at 4 and 10 mg upregulated trkA but only 10 mg P(4) increased p75(NTR). Five micrograms of E(2) coinjected with P(4) at 1.5 and 4 mg increased trkA, while p75(NTR) receptor was upregulated when coinjected with P(4) at 1.5 to 10 mg. The ovariectomy caused a decrease in trkA receptors compared to proestrus rats, and these decreases were significant by 6 wk, but surprisingly p75(NTR) increased at 2 wk after ovariectomy. 17 beta-Estradiol increased trkA but not p75(NTR) receptors in DRG, whereas P(4) caused increases in both trkA and p75(NTR) in DRG. In addition, the combination of these steroid hormones had more effect on both receptors than either hormone alone. Thus, we concluded that high levels of female steroid hormones such as those due to pregnancy or hormonal replacement therapy could increase NGF receptor expression in DRG that carry more NGF to elevate the CGRP synthesis in these groups. We suggested that the regulation of NGF receptors by ovarian steroids may underlie steroidal regulation of other factors such as CGRP.     

Multiple Levels for Regulation of TrkA in PC12 Cells by Nerve Growth Factor

Abstract: TrkA is a receptor tyrosine kinase for nerve growth factor (NGF). Recent studies indicate that NGF regulates not only activation of trkA kinase but also expression of the trkA gene. To further define NGF actions on trkA, we examined binding and signaling through trkA after both short and long intervals of NGF treatment. Induction of tyrosine phosphorylation on gp140 ^trkA was rapidly followed by down-regulation of cell surface and total cellular gp140 ^trkA . At later intervals, increased expression of trkA was evident in increased mRNA and protein levels. At 7 days, there was increased binding to gp140 ^trkA and increased signaling through this receptor. NGF appears to regulate trkA at several levels. In neurons persistently exposed to NGF, maintenance of NGF signaling may require increased trkA gene expression.     

Nerve Growth Factor as a Mitogen for a Pancreatic Carcinoid Cell Line

Abstract: Carcinoid tumors are a group of neuroendocrine neoplasms distributed widely throughout the body but most commonly occurring in the gut. These tumors retain many characteristics of their neural crest origin, including secretion of neuroactive peptides and responsiveness to neurotrophic substances. Nerve growth factor (NGF), a neurotrophic protein involved in maintenance and differentiation of peripheral sympathetic and sensory neurons, regulates growth of several neural tumor cells by inducing a differentiated phenotype and subsequent inhibition of cell growth rate. We examined the actions of NGF in a functioning human pancreatic carcinoid cell line (termed BON). NGF has no effect on the cytoarchitecture or constitutive secretion of bioamines in this carcinoid cell line. NGF, however, stimulates the in vitro cellular proliferation of BON cells. BON cells possess mRNA for the NGF receptors (p75^LNGFR and p140^trkA) and membrane-associated tyrosine kinase activity is increased in response to NGF. Both the mitogenic activity of NGF, as well as the receptor-linked tyrosine kinase activity, can be abrogated in BON cells by the trkA inhibitor K-252a and specific anti-NGF antibody. Our studies demonstrate that NGF is a mitogen for this carcinoid cell line without effect on cellular phenotype or cytoarchitecture. NGF may play a role in the development and progression of human carcinoid tumors.     

Skin homeostasis during inflammation: a role for nerve growth factor

The skin is a neuroendocrine immune organ in which many different molecules operate in autocrine-paracrine manner to guarantee tissue homeostatsis in physiological and pathophysiological conditions. In this paper we examined NGF and p75 receptor expression in the skin, during CFA induced inflammation, in a time-course study. We also examined cutaneus innervation and proliferation, by means of immunohistochemistry and quantitative image analysis, RT-PCR and Western blot. Spontaneous and evoked pain-behavior was also measured in experimental rats. The main results can be summarized as follows: 1). a peripheral sensory neuropathy develops in this condition, as indicated by thermal hyperalgesia, thus leading to a sensory denervation of the hind-paw skin as indicated by disappearance of CGRP and PGP9.5-IR fibers; 2). NGF and p75 expression (mRNA and protein) increases in the skin (keratinocytes) in the acute phase of CFA inflammation; 3). at this stage, a higher proliferative activity is observed in the skin, as defined by the expression of cell cycle-associated protein Ki67; 4). in the long-lasting chronic phase there is a further up-regulation of NFG and p75 expression in the skin; 5). trkA mRNA expression inversely correlates with p75 and NGF mRNA expression. These results suggest that CFA chronic inflammation evolves from inflammation to a small fibers sensory neuropathy and NGF seems to play a role in both events.     

Differences in early and late responses between neurotrophin-stimulated trkA- and trkC-transfected SH-SY5Y neuroblastoma cells.

Despite their sympathetic neuroblast origin, highly malignant neuroblastoma tumors and derived cell lines have no or low expression of the neurotrophin receptor genes, trkA and trkC. Expression of exogenous trkA in neuroblastoma cells restores their ability to differentiate in response to nerve growth factor (NGF). Here we show that stable expression of trkC in SH-SY5Y neuroblastoma cells resulted in morphological and biochemical differentiation upon treatment with neurotrophin-3 (NT-3). To some extent, trkA- and trkC-transfected SH-SY5Y (SH-SY5Y/trkA and SH-SY5Y/trkC) cells resembled one another in terms of early signaling events and neuronal marker gene expression, but important differences were observed. Although induced Erk 1/2 and Akt/PKB phosphorylation was stronger in NT-3-stimulated SH-Y5Y/trkC cells, activation of the immediate-early genes tested was more prominent in NGF-treated SH-SY5Y/ trkA cells. In particular, c-fos was not induced in the SH-SY5Y/trkC cells. There were also phenotypic differences. The concentrations of norepinephrine, the major sympathetic neurotransmitter, and growth cone-located synaptophysin, a neurosecretory granule protein, were increased in NGF-treated SH-SY5Y/trkA but not in NT-3-treated SH-SY5Y/trkC cells. Our data suggest that NT-3/p145trkC and NGF/p140trkA signaling differ in some aspects in neuroblasoma cells, and that this may explain the phenotypic differences seen in the long-term neurotrophin-treated cells.