Expression of mRNAs for Neurotrophins (NGF,BDNF, and NT-3) and their Receptors (p75NGFR,Trk, TrkB,and TrkC) in Human Peripheral Neuropathies

The steady-state mRNA levels of NGF, BDNF and NT-3, and the mRNA levels of their receptors p75^NGFR, trk, trk,B and trkC were examined in various human peripheral neuropathies, to determine the correlation with myelinated fiber pathology and T cell and macrophage invasions in the diseased nerves. Steady state levels of p75^NGFR mRNAs were significantly elevated in nerves with axonal pathology. In contrast, steady state levels of trkB and trkC mRNA levels were diminished, trk mRNA was not detected in the human nerves. The NGF, BDNF, and NT-3 mRNA levels were elevated in the diseased nerves. The increase in BDNF and NT-3 mRNA levels were proportional to the extent of invasion of the nerves by T cells and macrophages, but did not directly correlate with axonal nor demyelinating pathology, thus suggesting that inflammatory cell invasions are involved in the regulation of BDNF and NT-3 mRNA expressions. These neurotrophin and their receptor gene expressions in the diseased human nerves would be regulated by an underlying pathology-related process, and could play a role in peripheral nerve repair.     

The nerve growth factor receptor: a multicomponent system that mediates the actions of the neurotrophin family of proteins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) are members of a family of structurally related proteins termed neurotrophins that promote the growth and survival of neurons in the central and peripheral nervous systems. Each of these proteins bind to at least two membrane receptors. One is the low affinity nerve growth factor receptor (p75), which binds each member of the neurotrophin family. The other is one of a family of tyrosine kinase receptors —trkA binds only NGF, the relatedtrkB receptor binds BDNF and NT-3, andtrkC binds NT-3 alone. This article reviews kinetic and biochemical information on p75 and its relationship to thetrk gene products.     

Neuroprotection by NGF and BDNF Against Neurotoxin-Exerted Apoptotic Death in Neural Stem Cells Are Mediated Through Trk Receptors,Activating PI3-Kinase and MAPK Pathways

Neural stem cells (NSC) undergo apoptotic cell death during development of nervous system and in adult. However, little is known about the biochemical regulation of neuroprotection by neurotrophin in these cells. In this report, we demonstrate that Staurosporine (STS) and Etoposide (ETS) induced apoptotic cell death of NSC by a mechanism requiring Caspase 3 activation, poly (ADP-ribose) polymerase and Lamin A/C cleavage. Although C17.2 cells revealed higher mRNA level of p75 neurotrophin receptor (p75^NTR) compared with TrkA or TrkB receptor, neuroprotective effect of both nerve growth factor (NGF) and brain-derived growth factor (BDNF) mediated through the activation of tropomyosin receptor kinase (Trk) receptors. Moreover, both NGF and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathway. Inhibition of Trk receptor by K252a reduced PARP cleavage as well as cell viability, whereas inhibition of p75^NTR did not affect the effect of neurotrophin on neurotoxic insults. Thus our studies indicate that the protective effect of NGF and BDNF in NSC against apoptotic stimuli is mediated by the PI3K/Akt and MAPK signaling pathway via Trk receptors. An erratum to this article can be found at     

Evidence for the participation of nerve growth factor and its low-affinity receptor (p75NTR) in the regulation of the myogenic program

We have studied expression and function of neurotrophins and their receptors during myogenic differentiation of C2C12 cells, a clonal cell line derived from mouse muscle that is capable of in vitro differentiation. The genes coding for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their common low-affinity receptor p75^{neurotrophin receptor} (p75^NTR) were shown to be expressed in C2C12 myoblasts and downregulated during myogenic differentiation and fusion into myotubes. Cocultures with dorsal root ganglia from day 8 chick embryos revealed neurite-promoting activities of C2C12 cells that ceased with myogenic differentiation. These data suggest a temporal and developmental window for the effect of myogenic cell-derived neurotrophins on neuronal as well as on myogenic cell populations. NGF was shown to increase DNA synthesis and cell growth of C2C12 myoblasts and to enhance myogenic differentiation in this cell line. We present evidence that NGF-mediated processes take place at stages preceding myogenic differentiation. Enhanced muscle differentiation was also seen in p75^NTR-overexpressing C2C12 myoblasts which maintained high levels of receptors but ceased to produce NGF during differentiation. In contrast, when exogenous NGF was present at the onset of myogenic differentiation of receptor-overexpressing cells, muscle cell development was strongly repressed. This indicates that downregulation of p75^NTR is necessary for guiding myogenic cells towards terminal differentiation. Since none of the trk high-affinity neurotrophin receptors could be demonstrated in C2C12 cells, we conclude that NGF mediates its nonneurotrophic effect via its low-affinity receptor in an autocrine fashion. J. Cell. Physiol. 176:10–21, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of Fas-induced apoptosis by p75 neurotrophin receptor in a human neuroblastoma cell line

Fas and p75 neurotrophin receptors (p75^NTR) are death receptors that alone induce apoptosis of SH-SY5Y neuroblastoma cell line respectively by Fas ligand or brain-derived neurotrophic factor (BDNF, a p75^NTR ligand). We report on the modulation of Fas-mediated apoptosis by concomitant p75^NTR activation. The exposure to both ligands suppressed the apoptotic effect. A co-localisation of Fas and p75^NTR receptors was evidenced by co-capping and immunoprecipitation assays. Moreover, a caspase-8 inhibitor suppressed the protective effect of the concomitant BDNF and Fas ligand stimulation, suggesting that p75^NTR and Fas receptors could share common signalling pathways.     

The p75 neurotrophin receptor

The low-affinity p75 molecule and trk tyrosine kinases serve as receptors for target-derived neurotrophins. While the mechanism by which receptor tyrosine kinases impart intracellular signaling has become well understood, the precise roles of the p75 receptor are not fully defined. The p75 neurotrophin receptor belongs to a family of transmembrane molecules which also serve as receptors for the tumor necrosis factor family of cytokines. Each receptor shares a common extracellular structure highlighted by conserved cysteine-rich repeats. Because NGF, BDNF, NT-3, and NT-4/5 bind to p75 with similar affinity, p75 may either act as a common subunit in a neurotrophin receptor complex with trk family members, or act by independent mechanisms to mediate biological actions of each neurotrophin. 1994 John Wiley & Sons, Inc.     

TRAF6 and p62 inhibit amyloid β-induced neuronal death through p75 neurotrophin receptor

Amyloid β (Aβ) aggregates are the primary component of senile plaques in Alzheimer disease (AD) patient’s brain. Aβ is known to bind p75 neurotrophin receptor (p75^NTR) and mediates Aβ-induced neuronal death. Recently, we showed that NGF leads to p75^NTR polyubiquitination, which promotes neuronal cell survival. Here, we demonstrate that Aβ stimulation impaired the p75^NTR polyubiquitination. TRAF6 and p62 are required for polyubiquitination of p75^NTR on NGF stimulation. Interestingly, we found that overexpression of TRAF6/p62 restored p75^NTR polyubiquitination upon Aβ/NGF treatment. Aβ significantly reduced NF-κB activity by attenuating the interaction of p75^NTR with IKKβ. p75^NTR increased NF-κB activity by recruiting TRAF6/p62, which thereby mediated cell survival. These findings indicate that TRAF6/p62 abrogated the Aβ-mediated inhibition of p75^NTR polyubiquitination and restored neuronal cell survival.     

TRAF6 and p62 inhibit amyloid β-induced neuronal death through p75 neurotrophin receptor

Amyloid β (Aβ) aggregates are the primary component of senile plaques in Alzheimer disease (AD) patient’s brain. Aβ is known to bind p75 neurotrophin receptor (p75^NTR) and mediates Aβ-induced neuronal death. Recently, we showed that NGF leads to p75^NTR polyubiquitination, which promotes neuronal cell survival. Here, we demonstrate that Aβ stimulation impaired the p75^NTR polyubiquitination. TRAF6 and p62 are required for polyubiquitination of p75^NTR on NGF stimulation. Interestingly, we found that overexpression of TRAF6/p62 restored p75^NTR polyubiquitination upon Aβ/NGF treatment. Aβ significantly reduced NF-κB activity by attenuating the interaction of p75^NTR with IKKβ. p75^NTR increased NF-κB activity by recruiting TRAF6/p62, which thereby mediated cell survival. These findings indicate that TRAF6/p62 abrogated the Aβ-mediated inhibition of p75^NTR polyubiquitination and restored neuronal cell survival.     

Bicaudal‐D1 regulates the intracellular sorting and signalling of neurotrophin receptors

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor‐containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain‐derived neurotrophic factor (BDNF)‐activated TrkB and p75 neurotrophin receptor (p75^NTR) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co‐localisation of these neurotrophin receptors with retromer‐associated sorting nexin 1. The resulting re‐routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling‐competent TrkB isoforms and p75^NTR available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand‐activated receptors.     

Neurotrophins and neurotrophin receptors in pulmonary sarcoidosis - granulomas as a source of expression

Background

Pulmonary sarcoidosis is an inflammatory disease, characterized by an accumulation of CD4⁺ lymphocytes and the formation of non-caseating epithelioid cell granulomas in the lungs. The disease either resolves spontaneously or develops into a chronic disease with fibrosis. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been suggested to be important mediators of inflammation and mediate tissue remodelling. In support of this, we have recently reported enhanced NGF levels in the airways of patients with pulmonary sarcoidosis. However, less is known about levels of BDNF and NT-3, and moreover, knowledge in the cellular sources of neurotrophins and the distribution of the corresponding neurotrophin receptors in airway tissue in sarcoidosis is lacking.

Methods

The concentrations of NGF, BDNF and NT-3 in bronchoalveolar lavage fluid (BALF) of 41 patients with newly diagnosed pulmonary sarcoidosis and 27 healthy controls were determined with ELISA. The localization of neurotrophins and neurotrophin receptors were examined by immunohistochemistry on transbronchial lung biopsies from sarcoidosis patients.

Results

The sarcoidosis patients showed significantly enhanced NT-3 and NGF levels in BALF, whereas BDNF was undetectable in both patients and controls. NT-3 levels in BALF were found higher in patients with non-Löfgren sarcoidosis as compared to patients with Löfgren''s syndrome, and in more advanced disease stage. Epithelioid cells and multinucleated giant cells within the sarcoid granulomas showed marked immunoreactivity for NGF, BDNF and NT-3. Also, immunoreactivity for the neurotrophin receptor TrkA, TrkB and TrkC, was found within the granulomas. In addition, alveolar macrophages showed positive immunoreactivity for NGF, BDNF and NT-3 as well as for TrkA, TrkB and TrkC.

Conclusions

This study provides evidence of enhanced neurotrophin levels locally within the airways of patients with sarcoidosis. Findings suggest that sarcoid granuloma cells and alveolar macrophages are possible cellular sources of, as well as targets for, neurotrophins in the airways of these patients.     

A Pro-Nerve Growth Factor (proNGF) and NGF Binding Protein, α2-Macroglobulin,Differentially Regulates p75 and TrkA Receptors and Is Relevant to Neurodegeneration Ex Vivo and In Vivo

Nerve growth factor (NGF) is generated from a precursor, proNGF, that is proteolytically processed. NGF preferentially binds a trophic tyrosine kinase receptor, TrkA, while proNGF binds a neurotrophin receptor (NTR), p75^NTR, that can have neurotoxic activity. Previously, we along with others showed that the soluble protein α₂-macroglobulin (α₂M) is neurotoxic. Toxicity is due in part to α₂M binding to NGF and inhibiting trophic activity, presumably by preventing NGF binding to TrkA. However, the mechanisms remained unclear. Here, we show ex vivo and in vivo three mechanisms for α₂M neurotoxicity. First, unexpectedly the α₂M-NGF complexes do bind TrkA receptors but do not induce TrkA dimerization or activation, resulting in deficient trophic support. Second, α₂M makes stable complexes with proNGF, conveying resistance to proteolysis that results in more proNGF and less NGF. Third, α₂M-proNGF complexes bind p75^NTR and are more potent agonists than free proNGF, inducing tumor necrosis factor alpha (TNF-α) production. Hence, α₂M regulates proNGF/p75^NTR positively and mature NGF/TrkA negatively, causing neuronal death ex vivo. These three mechanisms are operative in vivo, and α₂M causes neurodegeneration in a p75^NTR- and proNGF-dependent manner. α₂M could be exploited as a therapeutic target, or as a modifier of neurotrophin signals.     

Nerve growth factor–induced growth of sympathetic axons into the optic tract of mature mice is enhanced by an absence of p75NTR expression

Postganglionic sympathetic axons display a remarkable ability for new collateral growth in response to local increases in nerve growth factor (NGF). Elevating NGF levels within the brain also induces the directional growth of sympathetic axons, but not within myelinated pathways of adult mammals. In this investigation, we provide in vivo evidence that sympathetic axons are capable of NGF‐induced collateral growth through the microenvironment of mature myelinated pathways, especially in the absence of the p75 neurotrophin receptor (NTR). In transgenic mice overexpressing NGF centrally and expressing p75^NTR, only a few varicose sympathetic axons invade the optic tract after the first month of postnatal life. In other transgenic mice overexpressing NGF centrally but lacking p75^NTR expression, the incidence of sympathetic axons within this myelinated tract substantially increases. Moreover, numerous unmyelinated sympathetic axons cluster together to form large processes extending through the optic tract; such structures are first seen 8 weeks after birth. Only these large axon bundles display prominent immunostaining for GAP‐43, which is preferentially localized to the sympathetic fibers, since nonmyelinating Schwann cells are not associated with these axon bundles. These data provide the first direct evidence that sympathetic axons are indeed capable of NGF‐induced collateral growth into myelinated tracts of mature mammals, and that their continued growth through this microenvironment is markedly enhanced by the absence of p75^NTR expression. We propose that p75^NTR among sympathetic axons may either directly or indirectly limit collateral branching of these fibers in response to increased levels of NGF. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 51–66, 1999     

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: Comparison with the effects of the neurotrophins

We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3). All of these factors were retrogradely transported to sub-populations of sensory neuron cell bodies in the L4/L5 DRG of neonatal rats. The size distribution of ¹²⁵I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 μm²). ¹²⁵I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 μm². Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 μm², respectively. A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo. GDNF and NGF rescued nearly 100% of the sensory neurons. BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport. We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats. GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not. The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model. The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 22–32, 1997.     

p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75^NTR), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75^NTR and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75^NTR and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75^NTR/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75^NTR or TrkA. Interestingly, immunoreactivity to anti-p75^NTR antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75^NTR, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75^NTR is turned on.     

Dose and age‐dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptor

Nerve growth factor (NGF) and related neurotrophins are target‐derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF‐dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax^−/− mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA‐positive, and TrkA^−/− TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75^NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose‐ and age‐dependent fashion, mediated by p75^NTR signaling through TrkA expressing axons. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

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     

p75NTR Antagonistic Cyclic Peptide Decreases the Size of β Amyloid-Induced Brain Inflammation

Amyloid beta (Aβ) was shown to bind the 75 kD neurotrophin receptor (p75^NTR) to induce neuronal death. We synthesized a p75^NTR antagonistic peptide (CATDIKGAEC) that contains the KGA motif that is present in the toxic part of Aβ and closely resembles the binding site of NGF for p75^NTR. In vivo injections of Aβ into the cerebral cortex of B57BL/6 mice together with the peptide produced significantly less inflammation than simultaneous injections of Aβ and a control (CKETIADGAC, scrambled) peptide injected into the contralateral cortex. These data suggest that blocking the binding of Aβ to p75^NTR may reduce neuronal loss in Alzheimer’s disease.     

Expression and function of Xenopus laevis p75NTR suggest evolution of developmental regulatory mechanisms

Neurotrophins signal through two different classes of receptors, members of the trk family of receptor tyrosine kinases, and p75 neurotrophin receptor (p75^NTR), a member of the tumor necrosis factor receptor family. While neurotrophin binding to trks results in, among other things, increased cell survival, p75^NTR has enigmatically been implicated in promoting both survival and cell death. Which of these two signals p75^NTR imparts depends on the specific cellular context. Xenopus laevis is an excellent system in which to study p75^NTR function in vivo because of its amenability to experimental manipulation. We therefore cloned partial cDNAs of two p75^NTR genes from Xenopus, which we have termed p75^NTRa and p75^NTRb. We then cloned two different cDNAs, both of which encompass the full coding region of p75^NTRa. Early in development both p75^NTRa and p75^NTRb are expressed in developing cranial ganglia and presumptive spinal sensory neurons, similar to what is observed in other species. Later, p75^NTRa expression largely continues to parallel p75^NTR expression in other species. However, Xenopus p75^NTRa is additionally expressed in the neuroepithelium of the anterior telencephalon, all layers of the retina including the photoreceptor layer, and functioning axial skeletal muscle. Finally, misexpression of full length p75^NTR and each of two truncated mutants in developing retina reveal that p75^NTR probably signals for cell survival in this system. This result contrasts with the reported role of p75^NTR in developing retinae of other species, and the possible implications of this difference are discussed. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 79–98, 2001