Correlation of gp140 expression and NGF-induced neuroblast chemotaxis in the embryonic rat spinal cord

During rat embryogenesis, fibers containing nerve growth factor (NGF) are present near the target destinations of migratory spinal neuroblasts, suggesting that diffusible gradients of NGF provide signals to newly generated neurons in the developing cord. In vitro, pM concentrations of NGF induce neuroblast chemotaxis (directed migration along a chemical gradient), indicating evoked motility is mediated by high-affinity receptors. Binding of ¹²⁵I-labelled NGF to fetal cord cells provides additional evidence that rat spinal neuroblasts express the high-affinity receptors; however, their presence has not been directly demonstrated. In the present study, we used immunocytochemistry to show that the high-affinity NGF receptor protein, gp140^trk (trk) is detectable in embryonic spinal tissue sections and in cord dissociates. Correlation of trk expression with NGF-induced chemotaxis revealed that both the receptor protein expression and functional responses to NGF develop along a ventro-dorsal gradient that parallels the in vivo pattern of neurogenesis and migration. Analysis of the temporal changes in trk immunoreactivity demonstrated that expression of gp140^trk is bimodal, possibly reflecting multiple effects of NGF during development. Chemotaxis to NGF was blocked by nM concentrations of the kinase inhibitor, K252a, suggesting that NGF stimulates motility via high-affinity receptors coupled to kinase activity. Elevated 3′,5′-cyclic adenosine monophosphate (cAMP) also attenuated NGF-induced chemotaxis, presenting preliminary evidence that protein kinase A (PKA) may regulate motility responses to NGF.     

p75 nerve growth factor receptor modulates p140trkA kinase activity,but not ligand internalization,in PC12 cells

The biological activity of nerve growth factor (NGF) has been shown to be mediated by the p140^trkA receptor tyrosine kinase, while the role of the p75 NGF receptor (p75^NGFR) is still unresolved. Here we have investigated the relative contribution of p140^trkA and p75^NGFR to early consequences of NGF binding: ligand internalization, p140^trkA autophosphorylation, and tyrosine phosphorylation of Shc, phospholipase C_γ-1 (PLC_γ-1), and extracellular signal-regulated kinases (ERKs). It was found that NGF internalization was neither prevented by blocking p140^trkA activity using the protein kinase inhibitors methylthioadenosine, staurosporine, and K-252a, nor by inhibiting NGF binding to p75^NGFR with antibodies. However, when NGF binding to p140^trkA was reduced by the use of a synthetic peptide corresponding to amino acids 36–53 of human p140^trkA, internalization of NGF was decreased. Thus, at least in PC12 cells, internalization appears to require binding of NGF to p140^trkA, but occurs irrespective of p140^trkA kinase activity and ligand occupancy of p75^NGFR. The NGF triple mutant Lys-32/Lys-34/Glu-35 to Ala, which has been demonstrated to bind to p140^trkA, but not to p75^NGFR, induced tyrosine phosphorylation more rapidly than wild-type NGF. Likewise, NGF-induced tyrosine phosphorylation was accelerated when NGF binding to p75^NGFR was prevented with REX-IgG. These findings indicate that NGF binding by p75^NGFR may modulate NGF-induced p140^trkA kinase activity. © 1994 Wiley-Liss, Inc.     

Expression of neurotrophins and the low-affinity NGF receptor in septal and hippocampal reaggregate cultures: local physiologic effects of NGF synthesized in the septal region.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of a family of trophic factors designated the neurotrophins, each of which can bind to the low-affinity NGF receptor (LNGFR). To investigate the mechanisms that regulate the expression of the neurotrophins and the LNGFR in the developing brain, we grew cells from the embryonic mouse septum and hippocampus in reaggregating cell culture and compared neurotrophin and LNGFR expression in developing reaggregates with that seen in the developing septum and hippocampus in situ. NGF, BDNF, NT-3 and LNGFR were each expressed in septal and hippocampal reaggregates as well as the native septum and hippocampus. Additionally, the temporal expression profiles observed in reaggregates were generally similar to those seen in the respective brain regions in situ. In order to determine whether NGF can modulate neurotrophin or LNGFR expression, reaggregates were cultured in the continual presence of either exogenous NGF or anti-NGF antibodies. NGF-treated septal cultures expressed twice the level of LNGFR mRNA as was seen in untreated septal cultures; on the other hand, septal cultures grown in the presence of anti-NGF antibodies, to neutralize endogenously synthesized NGF, displayed a 3-fold decrease in LNGFR mRNA expression compared to untreated cultures. No effects of NGF or anti-NGF were observed on LNGFR expression in hippocampal reaggregates, or on neurotrophin mRNA expression in either reaggregate type. These results suggest that regulatory mechanisms intrinsic to the septal and hippocampal regions control neurotrophin and LNGFR expression. NGF is likely to be one of these regulatory cues since it acts locally in septal reaggregates to control the developmental expression of LNGFR mRNA. The possible roles of locally synthesized NGF and other neurotrophins in the development of septal neurons are discussed.     

Expression of trk receptors in the developing and adult human central and peripheral nervous system

A family of tyrosine receptor kinases known collectively as trk receptors plays an essential role in signal transduction mediated by nerve growth factor and related neurotrophins. To localize the major trk receptors (trkA, B and C) in the developing and adult central (CNS) and peripheral (PNS) nervous system, we generated monoclonal antibodies (MAbs) to extracellular (MAbs E7, E13, E16, E21, E29) and intracellular (MAb I2) domains of human trkA fused to glutathione S-transferase. Several MAbs (E7, E13, E16) recognized glycosylated trkA (gp 140^trk and gp110^trk) in Western blots, one MAb (E7) recognized non-glycosylated (p80^trk) and glycosylated trkA in immunoprecipitation assays, and two MAbs (E13, E29) detected trkA on the cell surface of NIH3T3 cells transfected with a trkA cDNA. Although generated to trkA fusion proteins, this panel of MAbs also recognized trkB and trkC in flow cytometric studies of NIH3T3 cells transfected with trkB or trkC cDNAs. Thus, we used these pan-trk MAbs to probe selected regions of the CNS and PNS including the hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and dorsal root ganglion (DRG) to localize trkA, B, and C receptors in the developing and adult human nervous system. These studies showed that trk receptors are expressed primarily by neurons and are detectable very early in the developing hippocampus, cerebellum, spinal cord, and DRG. Although the distribution and intensity of trk immunoreactivity changed with the progressive maturation of the CNS and PNS, immunoreactive trk receptors were detected in neurons of the adult human hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and DRG. This first study of trk receptor proteins in the developing and adult human CNS and PNS documents the expression of these receptors in subsets of neurons throughout the developing and adult nervous system. Thus, although the expression of trk receptor proteins is developmentally regulated, the constitutive expression of these neurotrophin receptors by neurons in many regions of the adult human CNS and PNS implies that mature trk receptor-bearing neurons retain the ability to respond to neurotrophins long after terminal neuronal differentiation is complete. © 1995 Wiley-Liss, Inc.     

Expression of the trk gene family of neurotrophin receptors in prevertebral sympathetic ganglia

When the phenotype of neurons in pre- and paravertebral sympathetic ganglia are compared, there are marked differences in NGF dependence, neuropeptide content, connectivity and electrophysiological properties. The trophic interactions that induce these differences are currently poorly understood. One explanation is that prevertebral neurons receive a second neurotrophic signal, other than NGF, from their target of innervation. If this is the case, neurons in the prevertebral ganglia should express another neurotrophin receptor, in addition to the NGF receptor (trk A). To test this prediction, the level of expression of three neurotrophin receptors, trk A, trk B and trk C, were examined in one paravertebral sympathetic ganglia, the SCG, and two prevertebral ganglia, the celiac and superior mesenteric ganglia. It was found that mRNA encoding the full-length form of the trkB receptor was barely expressed in the SCG. Significantly higher levels of full-length trk B mRNA expression were found in the prevertebral ganglia. Ligands of the trkB receptor may, therefore, contribute to the differentiation and/or survival of some prevertebral sympathetic neurons.     

A potential interaction of p75 and trkA NGF receptors revealed by affinity crosslinking and immunoprecipitation

Nerve growth factor binds independently to two transmembrane receptors, the p75 neurotrophin receptor and the p140^trk (trkA) tyrosine kinase receptor, which are both co-expressed in the majority of neuronal cells that respond to NGF. Previous findings have suggested that appropriate co-expression of the two receptors gives rise to high affinity NGF binding sites and increased neurotrophin responsiveness; however, evidence demonstrating a direct interaction between the two receptors in cell lines has been lacking. Here we have utilized affinity crosslinking agents with ¹²⁵I-NGF to detect an association of trkA and p75 receptors in embryonic spinal cord and brain tissues enriched in the two receptors. Although multimeric complexes of trkA and p75 were not detected by affinity crosslinking, immunoprecipitation of cross-linked NGF-receptor complexes with trk-specific antibodies resulted in selective immunoprecipitation of crosslinked p75. Our results indicate that the trkA and p75 receptors can potentially interact, and that such an association may be responsible for the generation of high affinity NGF binding sites. © 1995 Wiley-Liss, Inc.     

Characterization of neurotrophin receptors by affinity crosslinking

Neurotrophic factors regulate the developmental survival and differentiation of specific neuronal populations. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of the nerve growth factor (NGF) protein family, also known as the neurotrophins. Insights into the different roles of neurotrophins can be gained by studying the expression of their functional receptors. Here we report the development of procedures for their radiolabeling and efficient crosslinking to specific cell-surface receptors. BDNF and NT-3 receptors in cell lines and tissue preparations expressing receptors for the 2 neurotrophins have been identified using this affinity crosslinking procedure. Like NGF, BDNF and NT-3 crosslinking to the low affinity NGF receptor (p75^NGFR) on PC12 cells. BDNF and NT-3 also crosslinked to cells expressing p145^trkB protein, producing an approximately 160 kD neurotrophin-receptor complex. Crosslinking of the 2 neurotrophins in vivo to specific trk family members in many areas of the central nervous system also produced a 160 kD receptor complex. However, in all brain regions a complex of approx. 100 kD could also be identified, all or most of which represents crosslinking to a truncated from of trkB. The broad distribution of BDNF and NT-3 receptors throughout the CNS suggests that neurotrophins may have yet unrecognized functions on specific neuronal populations. BDNF and NT-3 receptors were also found in brain areas in which the neurotrophins themselves are also synthesized, suggesting that beyond long-range trophic effects, these proteins may also act as autocrine or short-range paracrine regulators. © 1993 Wiley-Liss, Inc.     

Expression of neurotrophins and Trk receptors in the avian retina

Using the RNase protection assay, we have found that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are expressed in the avian retina during development. The expression peaks around embryonic days 12–15, with decreasing levels at later stages of development. Abundant levels of NGF and BDNF but low levels of NT-3 mRNA were found in the adult retina. We also found that light/darkness regulated the levels of NGF and BDNF mRNAs but not the levels of NT-3 mRNA in the 5-day-old chicken retina. It was demonstrated that NGF and BDNF mRNA levels were up-regulated by light exposure. The cellular localization of mRNA expression for the neurotrophins and neurotrophin receptors TrkA, TrkB, and TrkC in the retina was studied using in situ hybridization. The patterns of NGF and trkA mRNA expression were very similar and were localized to the external part of the inner nuclear layer on the border with the outer plexiform layer and corresponded to the localization of horizontal cells. NT-3 labeling was also found over the external part of the inner nuclear layer, whereas trkC mRNA was found over all layers in the retina. BDNF labeling was found over all layers in the retina, whereas TrkB labeling was intense over cells in the ganglion cell layer, which is in agreement with the response of ganglion cells to BDNF stimulation. Functional neurotrophin receptors were suggested by the response of retinal explants to neurotrophin stimulation. These data indicate that the neurotrophins play local roles in the retina that involve interactions between specific neuronal populations, which were identified by the localization of the Trk receptor expression. The data also suggest that NGF and BDNF expression is regulated by normal neuron usage in the retina. © 1996 Wiley-Liss, Inc.     

Dexamethasone induces TrkA and p75NTR immunoreactivity in the cerebral cortex and hippocampus

Nerve growth factor (NGF) plays a crucial role in synaptic plasticity during brain development and adulthood by activating a dual receptor system composed of TrkA and p75 (p75NTR) receptors. Exogenous NGF modulates the expression of both receptors. Little is known about the ability of endogenous NGF to regulate the expression of these receptors in basal forebrain cholinergic terminals. The ability of glucocorticoids to increase NGF expression in the hippocampus prompted us to investigate whether the synthetic glucocorticoid dexamethasone (DEX) increases TrkA and p75NTR expression in NGF-target cholinergic neurons in developing rats. We first examined the effect of DEX on NGF mRNA by in situ hybridization. DEX given systemically (0.5 mg/kg, sc) for 1 week to 7-day-old rats elicited an increase in NGF mRNA levels in the dentate gyrus of the hippocampus and superficial layers II and III of the cerebral cortex. Immunohistochemical analysis of p75NTR and TrkA levels revealed a dramatic increase in p75NTR immunoreactivity (IR) in both basal forebrain and hippocampus and TrkA IR in the hippocampus. Interestingly, in DEX-treated rats more axonal terminals were immunopositive for p75NTR in the hippocampus and cortex, suggesting an increase in p75NTR IR in cell bodies as well as in terminals. Our data indicate that the endogenously produced NGF elicits biological changes similar to those of the exogenously delivered NGF. We suggest that glucocorticoids might regulate and coordinate cholinergic neuronal maturation by increasing the biosynthesis of NGF.     

High-dose anabolic androgenic steroids modulate concentrations of nerve growth factor and expression of its low affinity receptor (p75-NGFr) in male rat brain

The effects of treatment with a high dose of nandrolone or testosterone on nerve growth factor (NGF) levels and NGF low-affinity receptor (p75-NGFr) distribution in the brain were analyzed. Nandrolone, subcutaneously injected in rats for several weeks, caused an increase of NGF levels in the hippocampus and septum and a decrease in the hypothalamus. The number of p75-NGFr-immunoreactive neurons and the p75-NGFr expression levels were reduced in the septum and vertical and horizontal Broca's bands. Testosterone injections caused an increase of NGF levels in the hippocampus, septum, and occipital cortex and induced an upregulation of p75-NGFr in the forebrain NGF target regions. This testosterone effect suggests that nandrolone and testosterone affect brain NGF target cells by a different mechanism(s). Nandrolone may interfere with NGF transport and/or utilization by forebrain neurons, causing an altered p75-NGFr expression and NGF accumulation as a consequence. Since NGF is known to maintain forebrain neurons and to regulate neurobehavioral functions, including memory, learning, and defensive behavior, it is possible to hypothesize that this neurotrophin may play a role in the mechanism of action of anabolic androgenic steroids (AAS) in the brain and be associated with endocrine and behavioral dysfunctions occurring due to AAS abuse. J. Neurosci. Res. 47:198–207, 1997. © 1997 Wiley-Liss, Inc.     

Distribution of neurotrophin receptors in human palatine tonsils: an immunohistochemical study

Increasing evidence indicates that nerve growth factor (NGF) exerts effects on cells of the immune system, but the possible immunomodulatory effect of other neurotrophins (brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3; and NT-4/5) has not been studied. Neurotrophins act on responsive cells by binding a low-affinity pan-neurotrophin receptor (p75), and more specific high-affinity receptors (gp140trkAA, gp145trkB and gp145trkC considered as preferred signaling transduction receptors for NGF, BDNF and NT-3, respectively). The expression of neurotrophin receptor proteins may be considered, therefore, as a potential indication of neurotrophin activity. In the present study we investigated the distribution of both types of neurotrophin receptors in the human palatine tonsils using immunohistochemical methods. In the follicular germinal centers both lymphocytes and follicular dendritic cells (FDC) displayed gp75 IR, but not IR for trk neurotrophin receptor proteins. gp140trkA-like IR and gp145trkC-like IR were encountered on paracortical interdigitating cells (PIC), and in the high endothelial venule cells. gp145trkB-like IR was found in a cell subpopulation which probably represented macrophages. Present results suggest that NGF, NT-3 and NT-4/5 may act in PIC and indirectly in lymphocytes, whereas BDNF and NT-4/5 could control macrophages. The role of p75 on lymphocytes and FDC and whether trk neurotrophin receptor proteins present in lymphoid tissues are functional receptors for neurotrophins remains to be elucidated.     

Trembler mouse Schwann cells in culture: Anomalies in the synthesis of lipids and proteins

Nerve growth factor (NGF) mRNA is widely distributed throughout peripheral and central rat tissues and throughout the human central nervous system. In the rat, high levels were found in cerebral cortex, hippocampus and thalamus/hypothalamus, medium levels in striatum and brainstem and low levels in cerebellum and spinal cord. The hippocampal levels did not change following the surgical transection of the septohippocampal pathway; similarly, the ibotenic acid-induced lesion of the nucleus basalis magnocellularis did not affect the amounts of NGF mRNA in the cerebral cortex. NGF mRNA was also present in high amounts in human cortex and hippocampus, with only low levels in septum/nucleus basalis magnocellularis, suggesting that NGF may also function as a retrograde trophic messenger in the human central nervous system. No evidence was obtained for an insufficient production of NGF mRNA in senile dementia of the Alzheimer type. In peripheral rat tissues, the highest concentrations of NGF mRNA were found in vas deferens, heart, sciatic nerve, submandibular gland and skin, with low levels in tissues such as trigeminal ganglion and pituitary gland.     

In situ hybridization detection of trka mRNA in brain: Distribution,colocalization with p75NGFR and up-regulation by nerve growth factor

In situ hybridization techniques were used to examine the distribution and the nerve growth factor (NGF) regulation of trkA mRNA in the adult rat brain in order to identify neurons in discrete regions of the brain that may be NGF responsive. In agreement with previous studies, trkA mRNA was detected within cells located in the medial septum (MS), diagonal band of Broca (DBB), and caudate. trkA mRNA was also detected in many other regions of the brain, including the nucleus basalis of Meynert, substantia innominata, paraventricular nucleus of the thalamus, interpeduncular nucleus, prepositus hypoglossal nucleus, vestibular nudei raphe obscuris, cochlear nucleus, sensory trigeminal nuclei, and gigantocellular as well as perigigantocellular neurons in the medullary reticular formation. By combining in situ hybridization detection of trkA mRNA with immunocytochemical detection ofp75^NGFR, it was determined that the vast majority (> 90%) of the trkA mRNA-containing cells detected in the MS and DBB also express p75^NGFR. Likewise, the vast majority of p75^NGFR-IR cells detected in the MS and DBB expressed trkA mRNA. Intracerebroventricular infusions of NGF into the third ventricle adjacent to the preoptic area resulted in a 58% increase in relative cellular levels of trkA mRNA in the horizontal limb of the DBB. These data provide evidence that both p75^NGFR and trkA are expressed by NGF-responsive neurons in the MS and DBB. In addition, we note that areas that contained trkA mRNA and that also have been reported to contain p75^NGFR are areas where high-affinity NGF binding sites have been observed autoradio-graphically, whereas areas that contain either trkA or p75^NGFR alone are areas where no high-affinity NGF binding has been reported. Together, these findings suggest that both trkA and p75^NGFR play an important role in the formation of high-affinity NGF receptors in brain and, furthermore, suggest that NGF may have physiological effects within many regions of the brain outside of the basal forebrain.     

Expression of NGF and NGF receptor mRNAs in the developing brain: Evidence for local delivery and action of NGF

Nerve growth factor (NGF) is a well-documented target-derived trophic factor in the peripheral nervous system. Recently, proteins as well as mRNAs for both NGF and its receptor (NGF-R) have been detected in diverse areas in the central nervous system (CNS). Considerable evidence suggests that NGF also functions in the target synthesis/retrograde transport mode in the brain. For example, NGF is synthesized in the target hippocampus, as indicated by the presence of NGF message, and interacts with the receptors on terminals projecting from basal forebrain, where receptor mRNA is detectable. Spatial separation of NGF and receptor gene expression is consistent with the target mechanism of action. To ascertain whether local action may also occur in the CNS, we used sensitive nuclease protection assays to study the relationship of NGF and NGF-R expression in the developing brain. Our results indicate that in some brain areas, such as diencephalon, postnatal hippocampus, and olfactory bulb, NGF message was highly expressed, while receptor mRNA was virtually undetectable, suggesting that these areas serve as target sources of NGF for distant afferent neurons. By contrast, in other brain areas, such as cerebellum, striatum, perinatal olfactory bulb, and prenatal hippocampus, NGF and NGF-R mRNAs were coexpressed and coregulated developmentally. Consequently, local delivery and action of the trophic molecule may occur in these areas during these periods. We tentatively conclude that NGF may act through both distant and local modes in the developing CNS.     

Transient and persistent expression of NT-3/HDNF mRNA in the rat brain during postnatal development

Neurotrophin-3 (NT-3) is closely related to two known neurotrophic agents, NGF and brain-derived neurotrophic factor (BDNF), and acts upon overlapping, yet distinct, populations of peripheral ganglia. NT-3 mRNA expression in the adult rat brain is largely confined to the hippocampus. In this study, we have used in situ hybridization to examine expression of this novel neurotrophic factor during postnatal development. The striking observation was made that NT-3 mRNA was transiently expressed at high levels in the cingulate cortex during the first 2 weeks of age. In the hippocampus, the adult pattern of expression, in the CA2, medial CA1, and granule layer of the dentate gyrus, was detected at all ages examined. However, there were two major differences in NT-3 mRNA expression in the developing hippocampus: Labeled cells were detected in the hilar region of the dentate gyrus at postnatal day 1 (P1) and 1 week that were absent by 2 weeks of age. Further, the caudal hippocampus, which has a lower intensity of labeling than the rostral region in the adult, was devoid of NT-3-expressing cells in the P1 and 1-week-old rat brain. These data indicate a substantial plasticity in NT-3 mRNA expression and suggest that the spectrum of neurons supported by NT-3 during development is partially different from that in the mature rat brain.     

Differential Regulation of Nerve Growth Factor (NGF) Synthesis in Neurons and Astrocytes by Glucocorticoid Hormones

Glucocorticoid hormones are important regulators of brain development and ageing. Here we show that dexamethasone, a synthetic glucocorticoid, differentially affects the expression of nerve growth factor (NGF) in cultured neurons and astrocytes. Dexamethasone increased the levels of NGF mRNA in cultured hippocampal neurons in a time- and concentration-dependent manner, whereas it down-regulated the NGF mRNA levels in astrocytes. However, dexamethasone had no effect on the mRNA levels of brain-derived neurotrophic factor in the hippocampal neurons. Aldosterone, a mineralocorticoid, in higher concentrations also up-regulated NGF mRNA levels in the hippocampal neurons. Dexamethasone increased the levels of NGF mRNA in the rat hippocampus in vivo , but not to the same extent as observed with kainic acid, a glutamate receptor agonist. There is no apparent diurnal rhythm in the hippocampal NGF protein levels corresponding to circadian variations in the levels of glucocorticoid hormones in serum. The increase in NGF mRNA in the hippocampus in vivo following dexamethasone treatments may reflect the physiological response of hippocampal neurons to high glucocorticoid levels reached under conditions of stress.