Rita Levi-Montalcini and the discovery of NGF, the first nerve cell growth factor

The nerve growth factor (NGF) is a signaling protein, discovered by Rita Levi-Montalcini in the early 1950's for its effect on growth and differentiation of specific populations of neurons of the peripheral nervous system. Originally identified as neurite outgrowth-stimulating factor, later studies revealed that the purified molecule has a number of target cells in the central nervous system and on nonneuronal cells. Moreover, recent studies showed the potential therapeutic properties of NGF in neuropathies of the central and peripheral nervous system and diseases of the eye and skin. Here I briefly describe the discovery of NGF, the early studies of Rita LeviMontalcini, a pioneer in modern neuroscience, and my scientific and human experience working in her laboratory for over 40 years.     

NGF 对大鼠胚胎中脑神经细胞体外增殖和分化的影响

目的:探讨神经生长因子(nerve growth factor,NGF)对大鼠胚胎中脑神经细胞体外增殖和分化的影响。方法:在体外分离培养大鼠胚胎中脑神经细胞的培养液中加入不同浓度(10、50、100、200ng/ml)的NGF,培养不同时间,以不加神经营养因子的细胞为对照组,通过MTT法检测细胞活性,神经元特异性烯醇化酶免疫细胞荧光技术鉴定神经细胞,光镜下形态学观察各组大鼠中脑神经细胞体外增殖和分化情况。结果:胚胎中脑神经细胞胞体增大、突起延长且有丰富的神经纤维连结成网络状,细胞集落数增加,显示出剂量-效应关系。结论:一定剂量的NGF能促进大鼠中脑神经细胞分化和增殖,增强其活性。     

RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, ∼63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.     

NGF and the local control of nerve terminal growth

It is generally believed that the mechanism of action of neurotrophic factors involves uptake of neurotrophic factor by nerve terminals and retrograde transport through the axon and back to the cell body where the factor exerts its neurotrophic effect. This view originated with the observation almost 20 years ago that nerve growth factor (NGF) is retrogradely transported by sympathetic axons, arriving intact at the neuronal cell bodies in sympathetic ganglia. However, experiments using compartmented cultures of rat sympathetic neurons have shown that neurite growth is a local response of neurites to NGF locally applied to them which does not directly involve mechanisms in the cell body. Recently, several NGF-related neurotrophins have been identified, and several unrelated molecules have been shown to act as neurotrophic or differentiation factors for a variety of types of neurons in the peripheral and central nervous systems. It has become clear that knowledge of the mechanisms of action of these factors will be crucial to understanding neurodegenerative diseases and the development of treatments as well as the means to repair or minimize neuronal damage after spinal injury. The concepts derived from work with NGF suggest that the site of exposure of a neuron to a neurotrophic factor is important in determining its response. 1994 John Wiley & Sons, Inc.     

The neurotrophins and their receptors

The neurotrophins, which include nerve growth factor (NGF) and its relatives, were discovered and characterized for their distinctive ability to promote survival and differentiation of postmitotic neurons. Perhaps surprisingly, the neurotrophins have recently been found to utilize a family of receptor tyrosine kinases (the Trks) similar to those used by normally mitogenic growth factors. In fact, ectopic expression of the Trks in non-neuronal cells allows them to mediate conventional mitogenic responses to the neurotrophins. Despite similarities with other receptor tyrosine kinases, the Trks are rather unique in that they are almost exclusively expressed in the nervous system, and they also display a number of novel structural features. In addition to the Trks, the neurotrophins all bind to another cell surface receptor (known as p75 or the low-affinity NGF receptor), whose role remains quite controversial.     

N-Propionylmannosamine-induced over-expression and secretion of thioredoxin leads to neurite outgrowth of PC12 cells

The function of the central nervous system largely depends on growth and differentiation (neurite outgrowth) of neural cells and it is well established that growth factors, especially nerve growth factor NGF stimulate neurite outgrowth. However, additional factors are implicated in this process notably the redox state of the cells. For the first time we could demonstrate that the application of recombinant thioredoxin stimulates neurite outgrowth of PC12 cells to the same extend as NGF. Thioredoxin, a small redox protein is a major player in the cellular protein reduction system. An increased expression and secretion of thioredoxin is achieved by the application of the novel sialic acid precursor N-propionylmannosamine (ManNProp). From earlier studies it is known that this N-acylmannosamine analog stimulates significantly the neurite outgrowth in cell cultures. This finding would give new insights into the mechanism of the nerve-stimulatory action of ManNProp and demonstrates the novel role of thioredoxin during the regulation of nerve growth, encouraging further studies.     

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 10²10⁴ cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.Abbreviations Ab-IgG-FITC anti-mouse-IgG labeled with fluorescein isothiocyanate - Ab-NF monoclonal antibody to neurofilament 160 kD - Ab-NGFR monoclonal antibody to NGF receptor - BDNF brain-derived neurotrophic factor - D-medium medium for differentiation culture - DMEM Dulbecco's modified Eagle's medium - M-medium medium for multiplication culture - NGF nerve growth factor - NGFR NGF receptor - NT-3 neurotrophin-3 - PC12 pheochromocytoma cell line - PC12h-R subclone of PC12 - Sup-D supernatant of D-medium     

Neuronal differentiation signals are controlled by nerve growth factor receptor/Trk binding sites for SHC and PLC gamma.

Differentiation and survival of neuronal cell types requires the action of neurotrophic polypeptides such as nerve growth factor (NGF). In the central and peripheral nervous system and the phaeochromocytoma cell model PC12, NGF exerts its effects through the activation of the signalling capacity of Trk, a receptor tyrosine kinase (RTK) which upon interaction with NGF becomes phosphorylated on tyrosines and thereby acquires the potential to interact with signal-transducing proteins such as phospholipase C-gamma (PLC gamma), phosphatidylinositol-3'-kinase (PI3'-K) and SHC. Mutagenesis of the specific binding sites for these src homology 2 (SH2) domain-containing substrates within the Trk cytoplasmic domain suggests a non-essential function of PI3'-K and reveals a major role for the signal controlled by the SHC binding site at tyrosine 490 and a co-operative function of the PLC gamma-mediated pathway for neuronal differentiation of PC12 cells.     

神经生长因子及其受体相关信号传导通路的研究进展

神经生长因子是神经营养因子家族成员之一,对不同时期神经元的存活、分化、生长及损伤后的修复和再生都有着十分重要的作用。不仅在神经系统中,随着人类的其他正常和肿瘤组织中同样也检测得到了NGF,神经生长因子在各方面的应用也得到了重视并均已得到了证实。NGF功能的发挥离不开与其受体的结合,根据NGF表面糖蛋白与凝集素结合能力的不同,其受体可被分为高亲和力受体酪氨酸激酶A和低亲和力受体p75。Trk A与NGF结合后所介导的信号通路主要有:1MAPK通路;2PLC-γ通路;3PI3K/PKB通路。而p75与NGF结合介导的信号传导通路主要包括:1NF-κB通路;2JNK-p53-Bax凋亡通路;3神经酰胺通路。Trk A一般介导的是正性信号,如促进神经细胞生长、维持神经细胞的存活等;而p75既可促进神经细胞存活,也可诱导神经细胞凋亡,但以后者为主。当Trk A与p75同时表达时,Trk A可抑制p75诱导细胞凋亡,使受损神经细胞大量增殖,所以其生物学总效应是促进神经细胞的生长和存活。     

Borna disease virus persistent infection activates mitogen-activated protein kinase and blocks neuronal differentiation of PC12 cells

Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF.     

Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues

Nerve growth factor (NGF) is a polypeptide important for normal development of the nervous system and promotion of survival and differentiation of sensory and sympathetic neurons in culture. The cellular effects of NGF are mediated by a specific cell surface molecule, nerve growth factor receptor (NGF-R). In the present study we have used a monoclonal antibody against human NGF-R to examine, by the avidin-biotin-immunoperoxidase method, the receptor distribution in a wide range of normal tissues and in more than 200 malignant tumors. Our results show that (a) human NGF-R is expressed in the peripheral nervous system but not in any of the central nervous system areas tested; (b) NGF-R expression is not restricted to neural tissues but is also found in a number of normal epithelial, mesenchymal, and lymphoid tissues; (c) NGF-R expression changes during normal development; and (d) NGF-R expression in malignant tumors generally parallels its normal tissue distribution. Thus, NGF-R is detected in a proportion of neuroectoderm-derived tumors, carcinomas, and lymphomas, and also in a characteristic group of small round-cell tumors (Ewing's sarcomas and embryonal rhabdomyosarcomas). These findings suggest a normal regulatory role for NGF in both neuronal and non-neuronal cells and identify a range of human tumors in which the NGF/NGF-R system may contribute to the malignant phenotype.     

Nerve growth factor has both mitogenic and antimitogenic activity

The present study demonstrates that nerve growth factor (NGF) possesses both antimitogenic and mitogenic activities. To this end, we have employed clonal PC12 rat pheochromocytoma cells and two PC12 variant sublines, U2 and U7. When PC12 cells are exposed to NGF in culture media that are otherwise either permissive (15% serum) or restrictive (1% serum) for proliferation, neuronal differentiation occurs and mitosis ceases. Variant lines of PC12 cells have been selected that continue to proliferate in the presence of NGF in permissive medium but which nevertheless retain NGF receptors and certain NGF responses. In contrast to the parent PC12 cells, when such variants were exposed to NGF in growth-restrictive media, cell proliferation was markedly stimulated. The mitogenic activity of NGF was detectable at 0.1 ng/ml (4 pM) and was maximal at 3 ng/ml (100 pM). Possible contamination of the NGF preparation by epidermal growth factor (EGF) or mitogenic proteolytic enzymes was ruled out by the use of anti-EGF and diisopropylfluoro-phosphate, respectively. These findings show that NGF shares the capacity to stimulate cell division with a variety of other peptide hormones and suggest that the mitogenic activity of NGF could play a role in development of the peripheral nervous system as well as in promotion of in vivo growth of certain neural crest-derived neoplasms.     

Thrombopoietin inhibits nerve growth factor-induced neuronal differentiation and ERK signalling

Thrombopoietin (TPO), a hematopoietic growth factor regulating platelet production, and its receptor (TPOR) were recently shown to be expressed in the brain where they exert proapoptotic activity. Here we used PC12 cells, an established model of neuronal differentiation, to investigate the effects of TPO on neuronal survival and differentiation. These cells expressed TPOR mRNA. TPO increased cell death in neuronally differentiated PC12 cells but had no effect in undifferentiated cells. Surprisingly, TPO inhibited nerve growth factor (NGF)-induced differentiation of PC12 cells in a dose- and time-dependent manner. This inhibition was dependent on the activity of Janus kinase-2 (JAK2). Using phospho-kinase arrays and Western blot we found downregulation of the NGF-stimulated phosphorylation of the extracellular signal-regulated kinase p42ERK by TPO with no effect on phosphorylation of Akt or stress kinases. NGF-induced phosphorylation of ERK-activating kinases, MEK1/2 and C-RAF was also reduced by TPO while NGF-induced RAS activation was not attenuated by TPO treatment. In contrast to its inhibitory effects on NGF signalling, TPO had no effect on epidermal growth factor (EGF)-stimulated ERK phosphorylation or proliferation of PC12 cells. Our data indicate that TPO via activation of its receptor-bound JAK2 delays the NGF-dependent acquisition of neuronal phenotype and decreases neuronal survival by suppressing NGF-induced ERK activity.     

Development of a Cell-Based Assay to Assess Binding of the proNGF Prodomain to Sortilin

Sortilin was first identified based on its activity as part of intracellular protein sorting machinery. Recently, it was discovered that sortilin also acts as a cell surface receptor for the propeptide form of nerve growth factor (proNGF), progranulin, and neurotensin. The interaction of sortilin to these neurotrophic ligands is linked to diseases of the nervous system that lead to neurodegeneration and neuropathic pain. Blocking of the interaction of sortilin to these ligands may prevent or slow the progress of these nervous system disorders. In vitro screening assays for blocking compounds or peptides are part of the standard set of tools for drug discovery. However, assays for sortilin biology are not readily available to determine if the selected blocking agent inhibits sortilin activity on the surface of cells. We have developed a sortilin specific cell based assay to identify compounds that specifically block interaction between sortilin and proNGF prodomain. The assay system records both the presence of sortilin on the cell surface and the interaction with the pro domain of NGF. Fluorescent images of the sortilin expressing cells are analyzed for the presence of pro domain of NGF. Sortilin-positive and sortilin-negative cells within one well are concomitantly and automatically analyzed. Sortilin—pro domain interaction can be blocked dose dependently by neurotensin and synthetic compounds. The assay will facilitate the discovery of entities interfering with the binding of sortilin to the NGF pro domain. This assay can be modified to screen for inhibitors of the binding of ligands to other complex cell surface receptors.     

肌肉注射人神经生长因子基因逆轴突传递修复神经损伤的研究进展

神经生长因子(NGF)促进中枢及外周神经系统神经元细胞存活、分化、轴突再生等重要作用已得到临床的广泛证实。目前临床上主要以局部或肌肉注射NGF蛋白的方式对神经系统的损伤进行治疗。但NGF半衰期短、局部应用副作用大、费用昂贵、难以透过血脑屏障等缺点而限制临床应用。长期以来,科研工作者致力于寻求一种理想的途径或方法以克服这一缺陷。随着基因工程技术的飞速发展,研究人员发现通过骨骼肌肌肉注射途径,以非病毒载体介导外源的NGF基因体内表达并逆轴突传递到神经损伤部位,有望解决这一难题。本文将就NGF及受体的基本结构和特性、逆轴突传递的机制、非病毒载体结合骨骼肌肌肉注射的基因治疗等方面进行总结和阐述。     

Retinoic acid regulates both expression of the nerve growth factor receptor and sensitivity to nerve growth factor.

In PC12 cells, retinoic acid (RA) stimulates the expression of p75NGFR, a component of the nerve growth factor (NGF) receptor, as indicated by a rapid increase in p75NGFR mRNA, an increase in the binding of 125I-labeled NGF to p75NGFR, and an increase in the binding of NGF to low affinity sites. RA-treated cells are more sensitive to NGF, but not to either fibroblast growth factor or phorbol 12-myristate 13-acetate, showing that RA has a specific effect on the responsiveness of PC12 cells to NGF. Exposure to RA leads neither to an increase in the expression of mRNA for trk, another component of the NGF receptor, nor to an increase in binding to high affinity receptors, suggesting that an increase in the expression of p75NGFR is sufficient to make cells more sensitive to NGF. This work suggests that, in addition to having direct effects on gene expression, RA can indirectly modulate differentiation of neurons by modifying their expression of cell surface receptors to peptide growth factors.     

TGF-beta modulates programmed cell death in the retina of the developing chick embryo

Programmed cell death (PCD) is a key phenomenon in the regulation of cell number in multicellular organisms. We have shown that reduction of endogenous transforming growth factor beta (TGF-beta) prevents apoptotic PCD of neurons in the developing peripheral and central nervous system, suggesting that TGF-beta is an important mediator of ontogenetic neuron death. Previous studies suggested that there are other pro-apoptotic molecules, nerve growth factor (NGF) and brain-derived neurotrophic factor, that induce cell death in the nervous system. In the developing chick retina, NGF induces PCD by activation of the p75 receptor. We have studied the role of TGF-beta and its putative interdependence with NGF-mediated PCD in the chick retina. We found that TGF-beta is present in the developing chick retina during the period of PCD and is essentially required to regulate PCD of retinal cells. TGF-beta 2, TGF-beta 3 and the ligand-binding TGF-beta receptor can be detected immunocytochemically in the central retina, a region where apoptosis is most prominent during the early period of PCD. Application of a TGF-beta-neutralizing antibody to chick embryos in ovo resulted in a decrease in the number of TUNEL-positive cells and a reduction of free nucleosome levels. In terms of magnitude, reduction of PCD caused by the neutralization of endogenous TGF-beta was equivalent to that seen after anti-NGF application. Neutralization of both factors did not result in a further decrease in apoptosis, indicating that NGF and TGF-beta may act on the same cell population. Furthermore, neutralization of TGF-beta did not affect the expression of NGF or the p75-receptor. Our results suggest that TGF-beta and NGF are both required to regulate cell death in the chick retina in vivo.