In autocrine cells, both a ligand and its receptors are synthesized in the same cell, but their intracellular interaction is not well known. We examined it using PC84 cells, a mutant PC12 cell line expressing nerve growth factor (NGF). We have already reported that the intracellular precursor of TrkA was phosphorylated and that MAP kinase was phosphorylated in PC84 cells. In this paper we found that the NGF receptors, TrkA and p75NTR, existed mainly as precursors, and most p75NTR localized inside PC84 cells. The phosphorylation of MAP kinase was also observed even when PC84 cells were incubated with anti-NGF antibody to block the extracellular interaction. These results suggest the possibility that newly synthesized NGF could interact intracellularly with the receptors in PC84 cells.

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.

Nerve growth factor (NGF) is a member of the neurotrophic factor family and plays a vital role in the physiological processes of organisms, especially in the nervous system. Many recent studies have reported that NGF is also involved in the regulation of tumourigenesis by either promoting or suppressing tumor growth, which depends on the location and type of tumor. However, little is known regarding the effect of NGF on interspinal schwannoma (IS). In the present study, we aimed to explored whether mouse nerve growth factor (mNGF), which is widely used in the clinic, can influence the growth of interspinal schwannoma cells (ISCs) isolated from IS in vitro.

Approximately one half of the neurons in the lumbar dorsal root ganglion of adult rats display high-affinity receptors for nerve growth factor (NGF). To ascertain which types of sensory neurons are potentially responsive to NGF, adjacent cryostat sections of rat dorsal root ganglia were processed either for NGF-receptor using radioautography or by one of four histochemical procedures. Histograms of the densities of neuronal labelling by radioiodinated NGF were examined for subpopulations of lumbar sensory neurons with thiamine monophosphatase enzyme activity or with immunoreactivity for calcitonin gene-related peptide (CGRP), substance P, or somatostatin. Virtually all neurons with strong CGRP immunoreactivity had high-affinity NGF binding sites, although some neurons with faintly positive CGRP immunoreactivity lacked such NGF binding. A subpopulation of large neurons, approximately 5% of the total, had dense labelling by 125I-NGF but were not stained by this immunohistochemical technique for CGRP. Of the three major populations of small neurons those with substance P immunoreactivity were consistently and heavily labelled by radioiodinated NGF whereas those with somatostatin immunoreactivity or thiamine monophosphatase activity were not specifically labelled by radioautography. For these primary sensory neurons in mature rats the genes for substance P and CGRP seem to be strongly expressed only in neurons capable of responding to NGF. On the other hand, neurons containing somatostatin and thiamine monophosphatase invariably lack high-affinity NGF receptors.

Temporal and spatial localization of nerve growth factor receptor (p75NGFR) in the developing olfactory system and gonadotropin-releasing hormone-1 (GnRH) system was characterized and its role analyzed using p75NGFR null mice and nasal explants. Prenatally, p75NGFR was expressed by GnRH neurons and olfactory ensheathing cells (OECs). In p75NGFR null mice, no change in the number of GnRH cells was detected as compared to wild-type. However, in null mice, a shift in the distribution of GnRH neurons was found, with a small population of GnRH cells migrating further caudally toward the median eminence. Additionally, a reduction of both GAD67 positive olfactory axons and GFAP positive OEC fibers occurred. Acute administration of a p75NGFR blocker to GnRH cells maintained in vitro increased migration rate, consistent with the change in distribution detected in p75NGFR null mice. Chronic inhibition of p75NGFR caused an attenuation of olfactory axon fasciculation and a decrease in OEC density, again mimicking the changes detected in null mice. However, a reduction in GnRH cell number was found after chronic treatment that not observed in KO animals suggesting indirect changes occur during chronic treatment in vitro and/or a compensatory mechanism occurs in vivo that prevents loss of GnRH neurons in the absence of p75NGFR.

Lead is a potent developmental neurotoxicant that affects many aspects of cognition and behavior. The hippocampus and striatum are among the areas particularly sensitive to the effects of lead and cholinergic neurons in both regions depend upon nerve growth factor (NGF) for their survival and maturation. The present study examined the extent to which postnatal lead exposure may affect the survival and expression of neuroptrophin receptors of septo-hippocampal cholinergic projection neurons in the medial septum/vertical limb of the diagonal band of Broca (MS/VDB) and cholinergic neurons of the striatum. Weanling rats were fed chow containing lead acetate for 30 days and effects on cholinergic cell number and the number of cells expressing neurotrophin receptors p75(NGFR) and trkA were assessed. A decrease in the number of cells expressing p75(NGFR) and an increase in the number of cells expressing trkA receptor was observed in the MS/VDB of lead-exposed rats, without a loss of cholinergic cell number or alteration in cell size. Lead-exposure resulted in a significant decrease in trkA-expressing cells in the striatum but no change in the number or size of cholinergic neurons. These results suggest that a brief postnatal lead exposure does not result in loss of MS/VDB or striatal cholinergic neurons but does modify the expression of neurotrophin receptors in these regions. The significance of these effects on the septo-hippocampal and striatal functioning remains to be studied.

Vascular endothelial growth factor (VEGF) and endostatin are angiogenic and anti-angiogenic molecules, respectively, that have been implicated in neurogenesis and neuronal survival. Using alkaline phosphatase fusion proteins, we show that the PC12 neuronal cell line contains cell membrane receptors for VEGF but not for endostatin and the collagen XV endostatin homologue. Immunocytochemistry confirmed that proliferating and differentiated PC12 cells express VEGF receptors 1, 2 and neuropilin-1. While no functional effects of VEGF on PC12 cell proliferation and differentiation could be observed, a slight VEGF-induced reduction of caspase-3 activity in differentiated apoptotic PC12 cells was paralleled by transient activation of ERK1/2 and Akt. In direct comparison, nerve growth factor proved to be a strikingly more potent neuroprotective agent than VEGF.

We have previously demonstrated that high concentrations of nerve growth factor suppress neurite outgrowth from sensory neurons. Inhibition could be mediated by either the p75NTR or TrkA receptor. We used a functional block of p75NTR by REX antibody in rat dorsal root ganglion neurons and dorsal root ganglion cultures from p75NTR knockout mice. In both systems, high-dose NGF inhibited neurite outgrowth, implying that p75NTR is not involved in suppression of neurite outgrowth. Confocal images of dissociated dorsal root ganglion neurons exposed to fluorescence-tagged NGF showed ligand internalization. Radioligand binding indicated disappearance of high-affinity binding sites from the surface of dorsal root ganglia after treatment with 200 ng/ml NGF for 1 h. Downstream signaling showed sustained hyperphosphorylation of MAPK (Erk(1-2)) but not of SNT or Akt. High-dose NGF may induce cytoplasmic relocation of the receptor TrkA and axonal growth arrest independently of p75NTR.

A molecule identical to nerve growth factor, with ovulation-inducing properties has been discovered in the seminal plasma of South American camelids (ovulation-inducing factor/nerve growth factor; OIF/NGF). We hypothesize that the ovulatory effect of OIF/NGF is initiated at the level of the hypothalamus, presumably by GnRH neurons. The objective of the present study was to determine the structural relationship between GnRH neurons and neurons expressing high- and low-affinity receptors for NGF (i.e., TrkA and p75, respectively) in the hypothalamus.

Selective serotonin reuptake inhibitors (SSRIs) have been widely used and are a major therapeutic advance in psychopharmacology. However, their pharmacology is quite heterogeneous. The SSRI fluvoxamine, with sigma-1 receptor agonism, is shown to potentiate nerve-growth factor (NGF)-induced neurite outgrowth in PC 12 cells. However, the precise cellular and molecular mechanisms underlying potentiation by fluvoxamine are not fully understood. In this study, we examined the roles of cellular signaling pathways in the potentiation of NGF-induced neurite outgrowth by fluvoxamine and sigma-1 receptor agonists.

In skeletal muscle, free nerve endings are mostly located within the connective tissue. However, the distribution of sensory afferent fibres in healthy human masseter muscle tissues has not been studied.

Increased angiogenesis and an altered blood-brain barrier have been reported in the brain of dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy. To further elucidate the mechanisms underlying angiogenesis in Duchenne muscular dystrophy, in this study we evaluated whether nerve growth factor (NGF) and nerve growth factor receptors (NGFRs) are involved, then correlated NGF-NGFRs expression with vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) content and matrix metalloproteinases-2 and -9 (MMP-2 and -9) activity, by confocal laser microscopy and immunohistochemistry. Results showed that neurons, astrocytes and ependymal cells were strongly labeled by NGF in mdx brain, expressing NGFRs on glial and endothelial cells. In controls, NGF faintly labeled neurons and astrocytes, whereas endothelial cells were negative for NGFRs. Immunogold electron microscopy demonstrated NGFR gold particles on endothelial cells in mdx brain, while in controls few particles were recognizable only on glial end feet. Western blotting and real time polymerase chain reaction (RT-PCR) demonstrated a higher expression of NGF and NGFR mRNA and protein in mdx brain as compared to controls, and increase of VEGF-VEGFR-2 and active MMP-2 and -9 content. Overall, these data suggest that in the brain of mdx mice, an upregulation of the NGF-NGFRs system might be involved directly, or indirectly through the activation of VEGF-VEGFR-2 and MMP-2 and -9, in the angiogenic response taking place in this pathological condition.

Sympathetic neurons densely innervate the myocardium with non-random topology and establish structured contacts (i.e. neuro-cardiac junctions, NCJ) with cardiomyocytes, allowing synaptic intercellular communication. Establishment of heart innervation is regulated by molecular mediators released by myocardial cells. The mechanisms underlying maintenance of cardiac innervation in the fully developed heart, are, however, less clear. Notably, several cardiac diseases, primarily affecting cardiomyocytes, are associated with sympathetic denervation, supporting the hypothesis that retrograde 'cardiomyocyte-to-sympathetic neuron' communication is essential for heart cellular homeostasis. We aimed to determine whether cardiomyocytes provide nerve growth factor (NGF) to sympathetic neurons, and the role of the NCJ in supporting such retrograde neurotrophic signalling. Immunofluorescence on murine and human heart slices shows that NGF and its receptor, tropomyosin-receptor-kinase-A, accumulate, respectively, in the pre- and post-junctional sides of the NCJ. Confocal immunofluorescence, scanning ion conductance microscopy and molecular analyses, in co-cultures, demonstrate that cardiomyocytes feed NGF to sympathetic neurons, and that this mechanism requires a stable intercellular contact at the NCJ. Consistently, cardiac fibroblasts, devoid of NCJ, are unable to sustain SN viability. ELISA assay and competition binding experiments suggest that this depends on the NCJ being an insulated microenvironment, characterized by high [NGF]. In further support, real-time imaging of tropomyosin-receptor-kinase-A vesicle movements demonstrate that efficiency of neurotrophic signalling parallels the maturation of such structured intercellular contacts. Altogether, our results demonstrate the mechanisms which link sympathetic neuron survival to neurotrophin release by directly innervated cardiomyocytes, conceptualizing sympathetic neurons as cardiomyocyte-driven heart drivers. KEY POINTS: CMs are the cell source of nerve growth factor (NGF), required to sustain innervating cardiac SNs; NCJ is the place of the intimate liaison, between SNs and CMs, allowing on the one hand neurons to peremptorily control CM activity, and on the other, CMs to adequately sustain the contacting, ever-changing, neuronal actuators; alterations in NCJ integrity may compromise the efficiency of 'CM-to-SN' signalling, thus representing a potentially novel mechanism of sympathetic denervation in cardiac diseases.

Regenerative capability of the peripheral nervous system after injury is enhanced by Schwann cells (SCs) producing several growth factors. The clinical use of SCs in nerve regeneration strategies is hindered by the necessity of removing a healthy nerve to obtain the therapeutic cells. Adipose-derived stem cells (ASCs) can be chemically differentiated towards a SC-like phenotype (dASCs), and represent a promising alternative to SCs. Their physiology can be further modulated pharmacologically by targeting receptors for neurotransmitters such as acetylcholine (ACh). In this study, we compare the ability of rat dASCs and native SCs to produce NGF in vitro. We also evaluate the ability of muscarinic receptors, in particular the M2 subtype, to modulate NGF production and maturation from the precursor (proNGF) to the mature (mNGF) form. For the first time, we demonstrate that dASCs produce higher basal levels of proNGF and mature NGF compared to SCs. Moreover, muscarinic receptor activation, and in particular M2 subtype stimulation, modulates NGF production and maturation in both SCs and dASCs. Indeed, both cell types express both proNGF A and B isoforms, as well as mNGF. After M2 receptor stimulation, proNGF-B (25 kDa), which is involved in apoptotic processes, is strongly reduced at transcript and protein level. Thus, we demonstrate that dASCs possess a stronger neurotrophic potential compared to SCs. ACh, via M2 muscarinic receptors, contributes to the modulation and maturation of NGF, improving the regenerative properties of dASCs.

The angiogenic aspect of neurotrophins and their receptors rather than the neuroscientific aspect has been focused. However, their role in retinal vascular development is underdiscovered. The purpose of this study is to understand the role of neurotrophin receptors in retinal vascular development and the mechanisms of their action. To identify the expression of tropomyosin receptor kinase receptor (Trk) in developing retina, tissues of 4, 8, 12, 16 and 26 day-old mice were prepared for experiments. Immunohistochemistry and immunofluorescence double staining against glial fibrillary acidic protein and type IV collagen were performed. TrkA was expressed mainly along the vessel structure in inner part of retina, especially in retinal astrocyte. In cultured primary astrocyte, recombinant nerve growth factor (NGF) was used to activate TrkA. NGF induced the phosphorylation of TrkA, and it also enhanced the level of activated Akt and vascular endothelial growth factor (VEGF) mRNA. Inhibition of phosphoinositide 3-kinase (PI3K) reversed the NGF-induced activation of these two molecules. This study demonstrated that TrkA activation on NGF leads to VEGF elevation by PI3K-Akt pathway and therefore suggested that TrkA could be a stimulator of retinal vascular development.

Rivastigmine (Riv) is a potent and selective cholinesterase (acetylcholinesterase, AChE and butyrylcholinesterase, BuChE) inhibitor developed for the treatment of Alzheimer's disease (AD). To elucidate whether Riv causes neuronal differentiation, we examined its effect on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. At concentrations of 0-100 μM, Riv was non-toxic in PC12 cells. Riv caused dose-dependent (10-100 μM) enhancement of NGF-induced neurite outgrowth, which was completely inhibited by the TrkA antagonist GW-441756. By contrast, Riv-mediated enhancement of neurite outgrowth was not blocked by the acetylcholine receptor antagonists, scopolamine and hexamethonium. However, the sigma-1 receptor (Sig-1R) antagonist NE-100 and sigma-2 receptor (Sig-2R) antagonist SM-21 each blocked about half of the Riv-mediated enhancement of NGF-induced neurite outgrowth. Interestingly, the simultaneous application of NE-100 and SM-21 completely blocked the enhancement of NGF-induced neurite outgrowth by Riv. These findings suggest that both Sig-1R and Sig-2R play important roles in NGF-induced neurite outgrowth through TrkA and that Riv may contribute to neuronal repair via Sig-1R and Sig-2R in AD therapy.

Differentiation and function of pancreatic beta cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic beta cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in beta cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic beta-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

Nerve growth factor in the tear film and corneal epithelium is hypothesized to play an important role in ocular surface maintenance and corneal wound healing. The purpose of this study was to determine the expression of nerve growth factor and its high affinity (trkA) receptor in tears, cornea, and lacrimal glands of normal dogs, the modulation of nerve growth factor and its trkA receptor during corneal wound healing, and the effect of topical nerve growth factor application on canine corneal epithelial wound healing. In the first of three experiments, the nerve growth factor content of tears, corneal epithelium, lacrimal gland, and 3rd eyelid gland was determined in normal dogs by enzyme-linked immunosorbent assay and the expression of nerve growth factor and its trkA receptor were evaluated in the cornea and lacrimal glands by immunohistochemistry. In a second experiment, unilateral corneal epithelial defects were created, and tissues were evaluated for changes in nerve growth factor or trkA expression for 1 week. In a third experiment, bilateral corneal epithelial defects were created and the right eyes in each animal were treated 4 times daily with either recombinant human nerve growth factor, murine nerve growth factor, or nerve growth factor-blocking antibody. The results of this study showed that nerve growth factor levels in normal dog tears, corneal epithelium, third eyelid gland and lacrimal gland were 15.4+/-4.6 ng ml(-1), 33.5+/-12.3, 52.4+/-17.4 and 48.8+/-9.4 ng g(-1), respectively. NGF and trkA receptors were identified by immunohistochemistry in all tissues examined. After unilateral corneal wounding, nerve growth factor concentration increased in the tears bilaterally for 3 days, especially in the wounded eye, and then returned to pre-wounding values. Nerve growth factor content, and immunohistochemical staining for nerve growth factor and trkA, increased significantly in the ipsilateral cornea epithelium following unilateral wounding. Nerve growth factor concentrations in lacrimal and third eyelid glands also increased bilaterally (p<0.01) after unilateral wounding. Time to wound closure and rate of epithelial migration did not differ significantly between nerve growth factor-treated, nerve growth factor antibody-treated, and control eyes. In conclusion, nerve growth factor is present under resting physiologic conditions in normal canine tears, and nerve growth factor and its trkA receptor are present under resting conditions in normal canine corneal epithelium, lacrimal gland and third eyelid gland. Nerve growth factor is elevated in the tears, cornea, and lacrimal glands after corneal epithelial wounding; however, topical application of nerve growth factor, or its blocking antibody does not modulate corneal wound healing in the normal dog eye.

Signaling of SorCS receptors by proneurotrophin ligands regulates neuronal plasticity, induces apoptosis and is associated with mental disorders. The detailed structure of SorCS2 and its extracellular specificity are unresolved. Here we report crystal structures of the SorCS2-NGF complex and unliganded SorCS2 ectodomain, revealing cross-braced SorCS2 homodimers with two NGF dimers bound in a 2:4 stoichiometry. Five out of six SorCS2 domains directly contribute to dimer formation and a C-terminal membrane proximal unreported domain, with an RNA recognition motif fold, locks the dimer in an intermolecular head-to-tail interaction. The complex structure shows an altered SorCS2 conformation indicating substantial structural plasticity. Both NGF dimer chains interact exclusively with the top face of a SorCS2 β-propeller. Biophysical experiments reveal that NGF, proNGF, and proBDNF bind at this site on SorCS2. Taken together, our data reveal a structurally flexible SorCS2 receptor that employs the large β-propeller as a ligand binding platform.

Most pulpal afferent neurons have cytochemical features in common with the class of nociceptors that express neuropeptides and respond to NGF, while very few bind the plant lectin IB4, a widely used marker for the class of nociceptors that respond to the GDNF family of neurotrophic factors. The present study was undertaken to determine whether the GDNF receptor, GFRalpha-1, is expressed by pulpal afferents, and, further, to determine whether tooth injury evokes changes in expression of the GDNF and NGF receptors among pulpal afferents. The tracer, fluoro-gold (FG), was applied to shallow cavities in dentin of first and second maxillary molars. After 4 weeks, the molars of one side received a test injury consisting of a deeper cavity that exposed pulp horns. Animals were perfusion fixed 2 days later, and sections of the trigeminal ganglia were double immunostained with combinations of antibodies against GFRalpha-1, and TrkA. Under control conditions, GFRalpha-1 immunostaining was observed in 72% of neurons that projected to the molar pulp, TrkA in 78%, and immunostaining for both receptors was observed in 65% of pulpal afferents. Tooth injury evoked up-regulation of GFRalpha-1 expression (to 93%) and a slight down-regulation of TrkA expression (67%) among tooth afferents, while there was no discernable change in the proportion of pulpal afferents that expressed both TrkA and GFRalpha-1 (to 61%).