The response of motoneurons to neurotrophins

The ongoing search for neurotrophic factors for motoneurons has led to the identification of a number of molecules which regulate motoneuron survival and function. Among these factors, the neurotrophins brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5 but not nerve growth factor (NGF), can prevent embryonic and postnatal motoneuron cell death in a variety of experimental paradigms. Analysis of expression of p75, trkB and trkC—components of the neurotrophin receptors—supports a potential physiological role for these factors as muscle- and glial-derived trophic factors for motoneurons. However, the survival of motoneurons during embryonic development is not reduced in the absence of BDNF, NT-3 or NT-4, as revealed by gene knockout experiments. This points to the involvement of additional trophic factors in the regulation of embryonic and postnatal motoneuron survival. The purpose of this review is to bring together the often prophetic observations from earlier studies—prior to the identification and characterization of these neurotrophins—with more recent results. Special issue dedicated to Dr. Hans Thoenen.     

Hippocampal neurotrophin levels in a kainate model of temporal lobe epilepsy: a lack of correlation between brain-derived neurotrophic factor content and progression of aberrant dentate mossy fiber sprouting

A significant upregulation of neurotrophins particularly brain-derived neurotrophic factor (BDNF) is believed to be involved in the initiation of epileptogenic changes such as the aberrant axonal sprouting and synaptic reorganization in the injured hippocampus. However, it is unknown which of the neurotrophins are upregulated during the peak period of aberrant mossy fiber sprouting in the chronically injured hippocampus. We measured chronic changes in the levels of BDNF, nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the adult hippocampus using enzyme-linked immunosorbent assay (ELISA) after a unilateral intracerebroventricular administration of kainic acid (KA), a model of temporal lobe epilepsy. For comparison, neurotrophins were also measured from the control intact hippocampus. Further, to see the association between changes in neurotrophin levels and the progression of mossy fiber sprouting, chronic changes in the mossy fiber distribution within the dentate supragranular layer (DSGL) were quantified. In the KA-lesioned hippocampus, the neurotrophins BDNF and NGF were upregulated at 4 days post-lesion, in comparison to their levels in the intact hippocampus. However, the concentration of BDNF reached the baseline level at 45 days post-lesion and dramatically diminished at 120 days post-lesion. In contrast, the upregulation of NGF observed at 4 days post-lesion was sustained at both 45 days and 120 days post-lesion. The concentration of NT-3 was upregulated at 45 days post-lesion but remained comparable to baseline levels at 4 days and 120 days post-lesion. Interestingly, analysis of mossy fiber sprouting revealed that most of the aberrant sprouting in the lesioned hippocampus occurs between 45 days and 120 days post-lesion. Taken together, these results suggest that the period of robust mossy fiber sprouting does not correlate with the phase of post-lesion BDNF upregulation. Rather, it shows a relationship with the time of upregulation of neurotrophins NGF and NT-3.     

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.     

Characterization of glial cell line-derived neurotrophic factor family receptor alpha-1 in peripheral nerve Schwann cells

Glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-1 (GFRalpha-1) is a receptor component of GDNF that associates with and activates the tyrosine kinase receptor Ret. To further understand GDNF and its receptor system in the PNS, we first characterized the expression of GFRalpha-1 in bovine peripheral nerve in vivo. GFRalpha-1 immunoreactivity was localized adjacent to the outermost layer of myelin sheath, as well as in the endoneurium and axoplasm. In a fractionation study, GFRalpha-1 was recovered mostly in the soluble fraction, although a small amount was recovered in the membrane fraction. A substantial amount of GFRalpha-1 in the membrane fraction was extractable by detergent and alkaline conditions. To further clarify the expression of GFRalpha-1 in Schwann cells, we examined cultured rat Schwann cells and the Schwannoma cell line RT4. Schwann cells expressed GFRalpha-1 in both the soluble/cytosolic and membrane fractions, and the membrane form of GFRalpha-1 was expressed at the outer surface of the Schwann cell plasma membrane. We also confirmed the secretion of the soluble form of GFRalpha-1 from Schwannoma cells in a metabolic labeling experiment. These data contribute to our knowledge of the production, expression and functions of GFRalpha-1 in the PNS.     

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation

While the role of p75^NTR signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75^NTR on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75^NTR-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75^NTR in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75^NTR, as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75^NTR-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75^NTR signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.     

Distinct modulatory actions of TGF-β and LIF on neurotrophin-mediated survival of developing sensory neurons

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are important for the regulation of survival and differentiation of distinct, largely non-overlapping populations of embryonic sensory neurons. We show here that the multifunctional cytokine transforming growth factor-β (TGF-β) fails to maintain sensory neurons cultured from embryonic day (E) 8 chick dorsal root ganglia (DRG), although DRG neurons are immunoreactive for the TGF-β receptor type II, which is essential for TGF-β signaling. However, in combination with various concentrations of NT-3 and NT-4, but not NGF, TGF-β3 causes a further significant increase in neuron survival. In DRG cell cultures treated with NGF, NT-3, and NT-4, a neutralizing antibody to TGF-β decreases neuron survival suggesting that endogenous TGF-β in these cultures affects the efficacies of neurotrophins. Consistent with this notion and a modulatory role of TGF-β in neurotrophin functions is the observation that TGF-β2 and-β3 immunoreactivities and TGF-β3 mRNA are located in embryonic chick DRG in close association with neurons from E5 onwards. We also show that leukemia inhibitory factor (LIF) significantly decreases NGF-mediated DRG neuron survival. Together, these data indicate that actions and efficacies of neurotrophins are under distinct control by TGF-β and LIF in vitro, and possibly also in vivo. Special issue dedicated to Dr. Hans Thoenen.     

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.     

K-252b Potentiation of Neurotrophin-3 Is trkA Specific in Cells Lacking p75NTR

Abstract: K-252b potentiates the neurotrophic effects of neurotrophin-3 (NT-3) in primary cultures of rat central cholinergic and peripheral sensory neurons and in a rat pheochromocytoma PC12 cell line. The ligand and receptor specificity, and role of the low-affinity neurotrophin receptor (p75^NTR) in the potentiation response induced by K-252b, are unknown. To address the issues of ligand and receptor specificity of K-252b potentiation, we have examined neurotrophin-induced DNA synthesis ([³H]thymidine incorporation) in NIH3T3 cells expressing trkA, trkB, or trkC . Neither NT-3 nor K-252b alone could stimulate mitogenic activity in the trkA -overexpressing clone. However, coaddition of K-252b (EC₅₀ of ∼2 n M ) with 10–100 ng/ml NT-3 led to incorporation of [³H]thymidine in trkA expressing cells to a level induced by optimal concentrations of nerve growth factor (NGF). The K-252b- and NT-3-induced [³H]thymidine incorporation correlated with an increase in the tyrosine autophosphorylation of the trkA receptor as well as tyrosine phosphorylation of trk -associated phospholipase C-γ1 and SH2-containing proteins. K-252b did not potentiate submaximal doses of NGF, or maximal doses of brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5) in trkA -expressing cells. Furthermore, K-252b did not potentiate DNA synthesis by submaximal doses of BDNF, NT-4/5, or NT-3 in trkB - or trkC -expressing NIH3T3 cells, suggesting that the potentiation profile for K-252b was specific for NT-3 in trkA -expressing cells. We found no expression of p75^NTR in the trk -expressing NIH3T3 cells. This is the first demonstration that K-252b potentiates a trkA -mediated biological nonneuronal response by NT-3 that occurs independent of p75^NTR and appears to be both ligand and receptor specific.     

The p75 neurotrophin receptor is localized to primary cilia in adult murine hippocampal dentate gyrus granule cells

The densely ciliated granule cell layer of the adult murine hippocampal dentate gyrus is one of two sites of adult neurogenesis. The granule cells have already been proven to localize their SSTR3 (somatostatin receptor 3) receptors to their so-called primary cilia. Here we show for the first time that 70-90% of these cells in 7-18 months-old wild-type and 3×Tg-AD (Alzheimer disease transgenic) mice also load p75^NTR receptors into the structures containing SSTR3, i.e., their primary cilia. On the other hand, p75^NTR’s TrkA co-receptors were not localized to cilia but conventionally distributed throughout the cell surface. Significantly fewer cells (20-40%) in the hippocampal CA1 and CA3 regions and cerebral cortex have p75^NTR containing cilia. While we don’t know what the impact of the cilial localization of p75^NTR on dentate gyral adult neurogenesis and memory encoding might be, the cilia’s amyloid β-activatable p75^NTR receptors could be damaging or lethal to the hippocampal functioning of amyloid β-accumulating Alzheimer brain.     

Myelin-associated glycoprotein-mediated signaling in central nervous system pathophysiology

The myelin-associated glycoprotein (MAG) is a type I membrane-spanning protein expressed exclusively in oligoden drocytes and Schwann cells. It has two generally known pathophysiological roles in the central nervous system (CNS): maintenance of myelin integrity and inhibition of CNS axonal regeneration. The subtle CNS phenotype resulting from genetic ablation of MAG expression has made mechanistic analysis of its functional role in these difficult. However, the past few years have brought some major revelations, particularly in terms of mechanisms of MAG signaling through the Nogo-66 receptor (NgR) complex. Although apparently converging through NgR, a readily noticeable fact is that the neuronal growth inhibitory effect of MAG differs from that of Nogo-66. This may result from the influence of coreceptors in the form of gangliosides or from MAG-specific neuronal receptors such as NgR2. MAG has several other neuronal binding partners, and some of these may modulate its interaction with the NgR complex or downstream signaling. This article discusses new findings in MAG-forward and-reverse signaling and its role in CNS pathophysiology.     

Glial cell line-derived neurotrophic factor-induced signaling in Schwann cells

Glial cell line-derived neurotrophic factor (GDNF), a known survival factor for neurons, has recently been shown to stimulate the migration of Schwann cells (SCs) and to enhance myelination. GDNF exerts its biological effects by activating the Ret tyrosine kinase in the presence of glycosylphosphatidylinositol-linked receptor, GDNF family receptor (GFR) alpha1. In Ret-negative cells, the alternative transmembrane coreceptor is the 140-kDa isoform of neural cell adhesion molecule (NCAM) associated with a non-receptor tyrosine kinase Fyn. We confirmed that GDNF, GFRalpha1 and NCAM are expressed in neonatal rat SCs. We found that GDNF induces an increase in the partitioning of NCAM and heparan sulfate proteoglycan agrin into lipid rafts and that heparinase inhibits GDNF-signaling in SCs. In addition to activation of extracellular signal-regulated kinases, and phosphorylation of cAMP response element binding protein, we found that cAMP-dependent protein kinase A and protein kinase C are involved in GDNF-mediated signaling in SCs. Although GDNF did not promote the differentiation of purified SCs into the myelinating phenotype, it enhanced myelination in neuron-SC cocultures. We conclude that GDNF utilizes NCAM signaling pathways to regulate SC function prior to myelination and at early stages of myelin formation.     

Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR.

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TeNT HC). Morphological and kinetic analysis of TeNT HC retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT HC carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT HC and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor p75NTR. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.     

Postnatal roles of glial cell line-derived neurotrophic factor family members in nociceptors plasticity

The neurotrophin and glial cell line-derived neurotrophic factor (GDNF) family of growth factors have been extensively studied because of their proven ability to regulate development of the peripheral nervous system. The neurotrophin family,which includes nerve growth factor (NGF), NT-3, NT4/5 and BDNF, is also known for its ability to regulate the function of adult sensory neurons. Until recently, little was known concerning the role of the GNDF-family (that includes GDNF, artemin, neurturin and persephin) in adult sensory neuron function. Here we describe recent data that indicates that the GDNF family can regulate sensory neuron function, that some of its members are elevated in inflammatory pain models and that application of these growth factors produces pain in vivo. Finally we discuss how these two families of growth factors may converge on a single membrane receptor, TRPV 1, to produce long-lasting hyperalgesia.     

Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR

Whereas the proform of the nerve growth factor (proNGF) is crucial for eliminating superfluous cells during neuronal development it also promotes apoptosis following brain trauma and neuronal injury. The apoptotic signal is elicited upon formation of a trimeric receptor complex also containing the vps10p domain receptor sortilin and the neurotrophin receptor p75^NTR. However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay to monitor the interaction between fluorescently tagged sortilin and p75^NTR in live cells. The method is based on a standard fluorescent plate reader found in many biochemical laboratories and the results are evaluated using a microscopy-based quantified sensitized acceptor emission FRET approach making use of a pair of FRET standard constructs. As a result, the effect of proNGF on the interaction between sortilin and p75^NTR can be evaluated in live cells allowing for screening and selection of therapeutic compounds interfering with proNGF-induced cell death.     

The role of neurotrophins in the developing nervous system

Neurotrophins were originally identified by their ability to promote the survival of developing neurons. However, recent work on these proteins indicates that they may also influence the proliferation and differentiation of neuron progenitor cells and regular several differentiated traits of neurons throughout life. Moreover, the effects of neurotrophins on survival have turned out to be more complex than originally thought. Some neurons switch their survival requirements from one set of neurotrophins to another during development, and several neurotrophins may be involved in regulating the survival of a population of neurons at any one time. Much of our understanding of the developmental physiology of neurotrophins has come from studying neurons of the peripheral nervous system. Because these neurons and their progenitors are segregated into anatomically discrete sites, it has been possible to obtain these cell for in vitro experimental studies from the earliest stage of their development. The recent generation of mice having null mutations in the neurotrophin and neurotrophin receptor genes has opened up an unparalleled opportunity to assess the physiological relevance of the wealth of data obtained from these in vitro studies. Here I provide a chronological account of the effects of members of the NGF family of neurotrophins on cells of the neural lineage with special reference to the peripheral nervous system. 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.     

NGF augments the autophosphorylation of Ret via inhibition of ubiquitin-dependent degradation

Nerve growth factor (NGF) is required for the trophic maintenance of postnatal sympathetic neurons. A significant portion of the growth-promoting activity of NGF is from NGF-dependent phosphorylation of the heterologous receptor tyrosine kinase, Ret. We found that NGF applied selectively to distal axons of sympathetic neurons maintained in compartmentalized cultures activated Ret located in these distal axons. Inhibition of either proteasomal or lysosomal degradation pathways mimicked the effect of NGF on Ret activation. Likewise, NGF inhibited the degradation of Ret induced by glial cell line-derived neurotrophic factor-dependent activation, a process that requires ubiquitination and proteasomal degradation. NGF induced the accumulation of autophosphorylated Ret predominantly in the plasma membrane, in contrast to GDNF, which promoted the internalization of activated Ret. An accretion of monoubiquitinated, but not polyubiquitinated, Ret occurred in NGF-treated neurons, in contrast to glial cell line-derived neurotrophic factor that promoted the robust polyubiquitination of Ret. Thus, NGF stimulates Ret activity in mature sympathetic neurons by inhibiting the ongoing ubiquitin-mediated degradation of Ret before its internalization and polyubiquitination.     

P75NTR在绒山羊毛囊生长周期中的表达

实验旨在研究绒山羊（Capra hircus）毛囊生长相关基因的表达规律,为绒山羊分子育种提供参考。本实验采用RTPCR、组织免疫荧光、Western blot等方法,研究神经营养素受体P75NTR在辽宁绒山羊皮肤组织中的表达和分布情况。结果表明：在毛囊生长周期的三个时期,均监测到P75NTR mRNA及蛋白的存在,P75NTR的荧光信号在退行期要强于其他两个时期,且在退行期毛囊外根鞘细胞中检测到P75NTR的高表达。以上结果表明,P75NTRmRNA及蛋白的表达与毛囊生长周期变化有一定的相关性,P75NTR受体在绒山羊的毛囊周期性变化中发挥着重要的功能。     

Haloperidol and atypical antipsychotics share a same action of decreasing P75(NTR) mRNA levels in PC12 cells

P75(NTR) is a common neurotrophin receptor which binds all neurotrophins with similar affinities and has been shown to be capable of mediating programmed cell death. In this study, we investigated effects of the antipsychotic drugs (APDs) haloperidol, clozapine, quetiapine, and risperidone on p75(NTR) mRNA levels in PC12 cells. Haloperidol is a prototype of typical APDs, and the other three drugs are atypical APDs, which are effective in reducing negative symptoms and cognitive deficits of schizophrenia, cause less side effects, and are more tolerable compared to haloperidol. PC12 cells were cultured with various concentrations of haloperidol, clozapine, quetiapine, or risperidone, in their media. After culture for 48h, the cell viabilities and p75(NTR) mRNA levels were measured. It was shown that both haloperidol and the atypical APDs used in this study deceased p75(NTR) mRNA levels in PC12 cells in a dose dependent manner, while not affecting cell viabilities. In further experiments, doses that produced significant/greatest effects were chosen and provided in the culture media for various periods. Decreases in p75(NTR) mRNA levels were observed in cultures treated for 12h with quetiapine, 24h with clozapine or risperidone, or for 48h with haloperidol. These results suggest that both haloperidol and atypical APDs have the same action of decreasing p75(NTR) mRNA levels in PC12 cells. Although the underlying molecular mechanism of this action remains to be elucidated, this finding is particularly relevant given the neurodevelopmental deficits associated with schizophrenia and important roles of p75(NTR) in mediating cell death.