Immunohistochemical localization of nerve growth factor (NGF) and NGF receptor (NGF-R) in the developing first molar tooth of the rat

Nerve growth factor (NGF) is a well established target-derived trophic factor supporting sympathetic and sensory innervation in the peripheral tissues as well as cholinergic innervation in the brain. Despite its name, NGF may have broader biological functions early in development in a wide range of non-neuronal differentiating cells. The many effects of NGF are directly dependent on initial binding of NGF to specific plasma membrane receptors on target cells. Here we use immunohistochemical methods to show that NGF and its receptor (NGF-R) are localized in a variety of embryonic epithelial and mesenchymal cells in the rat developing molar tooth. Dental cells known to play important roles in morphogenesis and inductive tissue interactions show NGF-like reactivity. Thus, labelling is seen in epithelial preameloblasts and mesenchymal odontoblasts. We also show a transient expression of NGF-R in restricted parts of the dental epithelium (inner dental epithelium) and dental mesenchyme differentiating cells (post-mitotic, polarizing odontoblasts). The expression patterns of NGF are different to those of NGF-R during embryogenesis and this is illustrated in detail in the developing tooth. The histochemical findings reported here support the notion that NGF may have multiple roles during morphogenetic and cytodifferentiation events in the tooth.     

Serum contains a macromolecular effector promoting the synthesis of nerve growth factor (NGF) in L cells

Addition of serum to the culture medium of murine L cells increased both the cellular level of NGF mRNA and the secretion of mature factor. Stimulation of NGF production by the serum was dose-dependent and appeared mediated by some specific factor(s). After gel filtration chromatography of serum, most of the biological activity formed a major peak with an apparent MW of about 160 kDa. This promoting factor was sensitive to heat at neutral pH, but resisted after heating at pH4. An activity inducing NGF synthesis, and displaying a comparable thermal sensitivity was also detected in Cohn fraction IV of human or bovine plasma.     

Evidence for the presence of nerve growth factor (NGF) and NGF receptors in human testis

Summary The co-localization of arginine vasopressin-and enkephalin-like immunoreactivities in nerve cells of the rat paraventricular hypothalamic nucleus and adjacent areas was investigated by the simultaneous application of immuno--galactosidase staining and the peroxidase-antiperoxidase method to sections. Arginine vasopressin-like immunoreactive cells were stained blue with immuno--galactosidase staining and enkephalin-like immunoreactive cells brown with the peroxidase-antiperoxidase method. Double-labeled cells with overlap of blue and brown immunoreaction products were identified in the anterior, medial, and lateral parvocellular parts of the paraventricular hypothalamic nucleus as well as in the previously indicated posterior magnocellular part. Other regions that contained double-labeled cells were the lateral hypothalamic area, anterior hypothalamic nucleus, area between the lateral hypothalamic area and anterior hypothalamic nucleus, suprachiasmatic nucleus, and bed nucleus of the stria terminalis, medial division, posterolateral part. These findings suggest that nerve cells with both arginine vasopressin- and enkephalin-like immunoreactivities may be more actively involved in neuroendocrine regulation and neural transmission than previously considered. They may provide a morphological basis for an increase in enkephalin-like immunoreactivity within the anterior pituitary in cases of hemorrhagic shock which is presumably accompanied by arginine vasopressin hypersecretion.Abbreviations AH anterior hypothalamic nucleus - ap anterior parvocellular part of the paraventricular hypothalamic nucleus - BSTMPL bed nucleus of the stria terminalis, medial division, posterolateral part - dp dorsal parvocellular part of the paraventricular hypothalamic nucleus - f fornix - LH lateral hypothalamic area - lp lateral paryocellular part of the paraventricular hypothalamic nuclcus - mp medial parvocellular part of the paraventricular hypothalamic nucleus - MPA medial preoptic area - pm posterior magnocellular part of the paraventricular hypothalamic nucleus - pv periventricular part of the paraventricular hypothalamic nucleus - SC suprachiasmatic nucleus - Zi zona incerta     

Retrograde transport of nerve growth factor (NGF) in motoneurons of developing rats: assessment of potential neurotrophic effects

The potential functional significance of nerve growth factor (NGF) receptors in spinal motoneurons was studied in newborn rats. 125I-NGF was specifically retrogradely transported by motoneurons from their peripheral nerve terminals. This transport was blocked by an excess of unlabeled NGF but not by cytochrome c. 125I-cytochrome c was not transported. The monoclonal anti-rat NGF receptor antibody, but not a control antibody, was also transported. Despite this ability of motoneurons to transport NGF, treatment of newborn rats with this factor did not increase motoneuron size or synthesis of neurotransmitter enzymes and did not prevent cell death after axotomy. We conclude that NGF receptors of spinal motoneurons can bind, internalize, and retrogradely transport NGF. However, these receptors do not mediate the classic trophic effects of NGF.     

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

A two-site radio-immunoassay for βNGF 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.     

Expression and localization of nerve growth factor (NGF) in the testis of alpaca (llama pacos)

During alpaca testis development and spermatogenesis, nerve growth factor (NGF) may play an important role. The main aim of this study was to determine the expression and localization of NGF in the alpaca testis, and to discuss the important role of NGF in alpaca reproductive characteristics. Immunohistochemical staining technique and real-time PCR were used. The expression of NGF in the same cells one-month old (newborn) alpacas 12-month, and 24-month old alpacas showed significant differences (p < 0.05); 12- and 24-month old alpacas showed no significant differences (p > 0.05); NGF at different cell stages showed no significant differences (p > 0.05). It suggests that NGF may be involved in the regulation of spermatogenesis, which provides direct evidence for NGF action in the alpaca testis during postnatal development and spermatogenesis.     

Paracrine and autocrine functions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brain-derived endothelial cells

Brain-derived neurotrophic factor (BDNF) is expressed by endothelial cells. We investigated the characteristics of BDNF expression by brain-derived endothelial cells and tested the hypothesis that BDNF serves paracrine and autocrine functions affecting the vasculature of the central nervous system. In addition to expressing TrkB and p75NTR and BDNF under normoxic conditions, these cells increased their expression of BDNF under hypoxia. While the expression of TrkB is unaffected by hypoxia, TrkB exhibits a base-line phosphorylation under normoxic conditions and an increased phosphorylation when BDNF is added. TrkB phosphorylation is decreased when endogenous BDNF is sequestered by soluble TrkB. Exogenous BDNF elicits robust angiogenesis and survival in three-dimensional cultures of these endothelial cells, while sequestration of endogenous BDNF caused significant apoptosis. The effects of BDNF engagement of TrkB appears to be mediated via the phosphatidylinositol (PI) 3-kinase-Akt pathway. Modulation of BDNF levels directly correlate with Akt phosphorylation and inhibitors of PI 3-kinase abrogate the BDNF responses. BDNF-mediated effects on endothelial cell survival/apoptosis correlated directly with activation of caspase 3. These endothelial cells also express p75NTR and respond to its preferred ligand, pro-nerve growth factor (pro-NGF), by undergoing apoptosis. These data support a role for neurotrophins signaling in the dynamic maintenance/differentiation of central nervous system endothelia.     

Neurosteroid dehydroepiandrosterone interacts with nerve growth factor (NGF) receptors, preventing neuronal apoptosis

The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75(NTR) membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [(3)H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75(NTR) receptors (K(D): 7.4 ± 1.75 nM and 5.6 ± 0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75(NTR) receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75(NTR) receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.     

eIF4F complex disruption causes protein synthesis inhibition during hypoxia in nerve growth factor (NGF)-differentiated PC12 cells

Poor oxygenation (hypoxia) influences important physiological and pathological situations, including development, ischemia, stroke and cancer. Hypoxia induces protein synthesis inhibition that is primarily regulated at the level of initiation step. This regulation generally takes place at two stages, the phosphorylation of the subunit α of the eukaryotic initiation factor (eIF) 2 and the inhibition of the eIF4F complex availability by dephosphorylation of the inhibitory protein 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). The contribution of each of them is mainly dependent of the extent of the oxygen deprivation. We have evaluated the regulation of hypoxia-induced translation inhibition in nerve growth factor (NGF)-differentiated PC12 cells subjected to a low oxygen concentration (0.1%) at several times. Our findings indicate that protein synthesis inhibition occurs primarily by the disruption of eIF4F complex through 4E-BP1 dephosphorylation, which is produced by the inhibition of the mammalian target of rapamycin (mTOR) activity via the activation of REDD1 (regulated in development and DNA damage 1) protein in a hypoxia-inducible factor 1 (HIF1)-dependent manner, as well as the translocation of eIF4E to the nucleus. In addition, this mechanism is reinforced by the increase in 4E-BP1 levels, mainly at prolonged times of hypoxia.     

Potentiation of nerve growth factor(NGF)-mediated neurite outgrowth in high K+ medium is associated with increased binding of iodinated NGF in PC12 cells

Nerve growth factor(NGF)-mediated neurite outgrowth of PC12 pheochromocytoma cells was potentiated in medium containing high concentrations of extracellular K+. The binding of iodinated NGF to the cells was also enhanced by raising the concentration of K+ in medium up to 100 mM; the enhancement was saturated at 50 mM K+. Although the mechanism by which NGF-mediated neurite outgrowth is potentiated in high K+ medium remains to be largely unknown, high K+-induced alterations in the NGF binding are suggested to play a role in this phenomenon.     

Mitogenic effect of nerve growth factor (NGF) in LNCaP prostate adenocarcinoma cells: role of the high- and low-affinity NGF receptors

We have investigated the effect of nerve growth factor (NGF) in the androgen-dependent, prostate adenocarcinoma LNCaP cell line. Exposure of LNCaP cells to NGF resulted in a significant increase of cell proliferation. The effect was concentration dependent and equally present in serum- or charcoal-stripped serum-supplemented and serum-deprived conditions. The mitogenic action of NGF was accompanied by an enhanced expression of prostate-specific antigen (PSA) and resulted additive to the proliferative effect of dihydrotestosterone. The proliferative effect of NGF appeared to be mediated by the high-affinity NGF receptor, p140trka. Only p140trka, but not the low-affinity NGF receptor, p75LNGFR, was expressed in LNCaP cells; both the proliferative response and the phosphorylation of p140trka upon NGF treatment were prevented by the tyrosine kinase inhibitor K252a. LNCaP cells transiently transfected with the cDNA encoding for p75LNGFR appeared more sensitive to NGF, as demonstrated by the increased number of p75LNGFR-transfected LNCaP cells exposed for 72 h to NGF compared with wild LNCaP cultures. However, p75LNGFR-transfected LNCaP cells rapidly underwent apoptotic death when deprived of NGF. Our study demonstrates the physiological relevance of NGF in the regulation of prostate cell proliferation and the relative contribution of the high- and low-affinity NGF receptors in this control.     

Influence of nerve growth factor (NGF) on formation of synapses in newt embryonic neural tube]

Under the influence of the nerve growth factor (NGF) a general stimulation of the synthetic processes, especially protein synthesis, occurs in the neuroblasts. The morphological counterpart is an hypertrophy of the nucleoli and spiralization of nucleolonema, an increase of the number of ribosomes with the persistence of a specific organization for each stage of development, then the proliferation of the granular endoplasmic reticulum and the modification of the predominant shape of mitochondria, which becomes annular, their outer surface being largely increased. The nutrient stores are quickly exhausted, with an accelerated disintegration of yolk droplets. There is an obvious increase in the number of non-synaptic contacts (puncta adherentia) between processes of neuroblasts and neighbouring nervous structures, and also an acceleration of formation of typical synaptic zones, indicating that NGF stimulates the functional maturation of the nervous system.     

Steady-state polypeptide modulations associated with nerve growth factor (NGF)-induced terminal differentiation and NGF deprivation-induced apoptosis in human neuroblastoma cells.

Human neuroblastoma SH-SY5Y cells differentiate terminally in culture upon exposure to nerve growth factor (NGF) for 4-5 weeks. The neuronal phenotypic properties acquired in response to prolonged NGF treatment include morphological differentiation, cessation of mitotic activity, neuronal marker expression, increased membrane electrical potentials, and a survival dependence upon NGF for trophic support (Jensen, L.M. (1987) Dev. Biol. 120, 56-64). Thus, differentiated cultures survive indefinitely in the continued presence of NGF, however, withdrawal of NGF from differentiated cultures effects the loss of cellular viability within 3-6 days. Here, we show that death of differentiated SH-SY5Y cells caused by NGF deprivation is characteristic of apoptosis. To compare the differentiation promoting and the neurotrophic properties of NGF, whole SH-SY5Y cell extracts were analyzed by two-dimensional polyacrylamide gel electrophoresis using isoelectric focusing and nonequilibrium pH gradient electrophoresis gels in the first dimension. Steady-state levels of polypeptides extracted from whole-cell lysates of naive (untreated) cells, terminally differentiated cells, and NGF-deprived differentiated cells were compared. Over 1,000 spots from each were analyzed using computer-aided spot matching and densitometry. We noted 25 polypeptides that decreased during differentiation, including 15 that decreased by a factor of 10 or more. The levels of five polypeptides were induced from very low or undetectable levels in naive cells. Withdrawal of NGF from terminally differentiated cells produced alterations in steady-state protein patterns substantially distinct from those occurring during differentiation. While levels of most proteins do not appear affected early after NGF withdrawal, others rapidly return to levels comparable with those of the naive state and some changes occurring with differentiation are enhanced further upon NGF withdrawal. Three polypeptides were regulated uniquely by NGF withdrawal, including two that were induced, on average, 20- and 28-fold and another that was depressed more than 7-fold after NGF deprivation, before cell death. These data indicate that NGF elicits both constitutive and nonconstitutive changes in gene expression and suggest that the differentiation promoting and the neurotrophic properties of NGF correlate with the regulation of different gene products.     

Crystallization and characterization of the high molecular weight form of nerve growth factor (7 S NGF)

A high molecular weight form of nerve growth factor (7 S NGF) has been crystallized in two crystal forms from polyethylene glycol 4000 by the vapour diffusion technique. The orthorhombic form A belongs to the space group P2(1)2(1)2(1) and has cell dimensions of a = 95.6, b = 96.5 and c = 147.0 A. With synchrotron X-ray radiation, these crystals diffract to 2.8 A resolution. They contain an intact 7 S NGF complex in the asymmetric unit. The tetragonal form B, which grows at similar conditions to the A form, belongs to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = 97.4, b = 97.4 and c = 308.3 A. These crystals diffract to 3.6 A resolution and contain one 7 S complex per asymmetric unit. Native X-ray data have been collected to 3.3 A for the A form and to 5.0 A for the B form, both using synchrotron radiation.     

NGF mRNA expression in developing cutaneous epithelium related to innervation density

To determine if the initial level of NGF mRNA in developing cutaneous epithelium is correlated with its final innervation density, we measured the concentration of NGF mRNA in the epithelia of the maxillary, mandibular and ophthalmic territories of trigeminal ganglion in the embryonic mouse. At the onset of neuronal death in the ganglion there were marked differences in the concentration of NGF mRNA in these epithelia: the level was highest in the epithelium of the densely innervated maxillary territory, it was lower in the epithelium of the moderately innervated mandibular territory and was lowest in the epithelium of the sparsely innervated ophthalmic territory. These regional differences in the level of NGF mRNA during the early stages of target field innervation suggest that the level of NGF production in target field cells, rather than regional differences in the access of innervating neurons to NGF, governs the number of neurons that survive. Because the same percentage cell death occurs in each of the subsets of trigeminal neurons that innervate the maxillary, mandibular and ophthalmic territories, regional differences in NGF synthesis are not responsible for establishing differences in innervation density, rather they maintain differences that arise earlier in development.