Specific effects of neuregulin-1β on the communication between DRG neurons and skeletal muscle cells in vitro

The communication between primary afferent neuron and skeletal muscle (SKM) is one of the important factors on maintaining the structure and function of SKM cells. Neuregulin-1β (NRG-1β) signaling is essential for regulating synaptic neurotransmission. Here, we established a neuromuscular coculture model of dorsal root ganglion (DRG) sensory neurons and SKM cells to explore the nerve-muscle communication in the presence of exogenous NRG-1β. The expression of three distinct subtypes (TrkA, TrkB, and TrkC) of tyrosine kinase receptors was monitored for the phenotypical alterations of the neurons. The aggregation extent of acetylcholine receptor (AChR) represents the specific changes of SKM cells after NRG-1β incubation in this neuromuscular coculture model. The results showed that NRG-1β not only enhanced neurite outgrowth of DRG neurons but also increased the length and branches of SKM cells. NRG-1β treatment not only induced expression of all the three subtypes of Trk receptors in neurons but also promoted AChR aggregation on the surface of SKM cells. The effects of NRG-1β could be blocked by administration of ERK1/2 inhibitor PD98059, PI3K inhibitor LY294002, and JAK2 inhibitor AG490, respectively. These data imply that NRG-1β is essential for the nerve-muscle communication by enhancing growth and modifying phenotypes of the two different kinds of cells. The specific effects produced by NRG-1β add novel interpretation for nerve-muscle communication between sensory neurons and SKM cells.     

Neuronal Phenotype and Tyrosine Kinase Receptor Expression in Cocultures of Dorsal Root Ganglion and Skeletal Muscle Cells

The neuropeptide‐immunoreactive (IR) and neurofilament‐IR neurons are two major phenotypical classes in dorsal root ganglion (DRG). Tyrosine kinase receptor (Trk)A, TrkB, and TrkC are three members of the Trk family which may be relevant to neuronal phenotypes. Whether target skeletal muscle cells generate their expression remains unclear. Neurons containing substance P (SP), calcitonin gene‐related peptide (CGRP), neurofilament 200 (NF‐200), TrkA, TrkB, and TrkC were quantified using immunohistochemistry in rat DRG neuronal cultures and cocultures of DRG neurons and skeletal muscle cells. The percentage of NF‐200 and TrkC‐expressing neurons in cocultures of DRG neurons and skeletal muscle cells was significantly higher, 26.86% ± 3.17% (NF‐200) and 27.74% ± 3.63% (TrkC) compared with 20.92% ± 1.98% (NF‐200) and 16.70% ± 3.68% (TrkC) in DRG cultures; whereas the percentage of SP, CGRP, TrkA, and TrkB‐expressing neurons was not changed significantly by the addition of target skeletal muscle cells. Thus, target skeletal muscle cells may influence neurofilament‐phenotype and TrkC receptor but not neuropeptide‐phenotype and TrkA and TrkB receptors. Anat Rec, 2009. © 2008 Wiley‐Liss, Inc.     

Sortilin associates with Trk receptors to enhance anterograde transport and neurotrophin signaling

Binding of target-derived neurotrophins to Trk receptors at nerve terminals is required to stimulate neuronal survival, differentiation, innervation and synaptic plasticity. The distance between the soma and nerve terminal is great, making efficient anterograde Trk transport critical for Trk synaptic translocation and signaling. The mechanism responsible for this trafficking remains poorly understood. Here we show that the sorting receptor sortilin interacts with TrkA, TrkB and TrkC and enables their anterograde axonal transport, thereby enhancing neurotrophin signaling. Cultured DRG neurons lacking sortilin showed blunted MAP kinase signaling and reduced neurite outgrowth upon stimulation with NGF. Moreover, deficiency for sortilin markedly aggravated TrkA, TrkB and TrkC phenotypes present in p75(NTR) knockouts, and resulted in increased embryonic lethality and sympathetic neuropathy in mice heterozygous for TrkA. Our findings demonstrate a role for sortilin as an anterograde trafficking receptor for Trk and a positive modulator of neurotrophin-induced neuronal survival.     

Cellular localization of the Trk neurotrophin receptor family in human non-neuronal tissues.

We investigated the distribution of the high-affinity neurotrophin receptors TrkA, TrkB, and TrkC in a wide range of normal non-neuronal tissues of adult human by immunohistochemical methods. Trk immunoreactivity (IR) was detected at various levels in all tissues examined, except for the heart and liver. The gastric parietal cells showed strong TrkA and TrkC IR and all of the Trks had IR for the putative intestinal neuroendocrine cells. In the pancreas, TrkA and TrkC IR was detected in the sub-intralobular ducts, whereas TrkB IR was found specifically in the alpha-islet cells. The lymph node and spleen exhibited TrkB IR in monocytes/macrophages. The adrenal cortex showed selective TrkA IR with TrkC IR in the medulla. In the reproductive system, TrkA IR was detected in the prostatic epithelial cells, TrkC in the ovarian theca and granulosa cells, TrkA and TrkC in the secretory-phase endometrium, and TrkA in the mammary ducts. The kidney showed strong TrkA and TrkC IR in it tubules, but no Trk receptors were present in the glomeruli. In the skin, TrkA and TrkB/TrkC were present in the basal and granular layers of the epidermis, respectively.     

Dehydroepiandrosterone and neurotrophins favor axonal growth in a sensory neuron–keratinocyte coculture model

We have previously shown that axonal growth from a subset of sensory neurons was promoted by keratinocytes when the two cell types were co-cultured in a low calcium medium. This phenomenon involves the production of one or several diffusible factors. Here we show that the neuritogenic effect of keratinocytes was significantly reduced in the case of rat primary sensory dorsal root ganglion (DRG) neurons, or completely suppressed in the case of the sensory neuron cell line ND7-23, when the activity of neurotrophin receptors (Trk receptors) was blocked with K252a. This trophic effect apparently involved the activation of tyrosine kinase receptors A and B (TrkA and TrkB) expressed by subpopulations of small- to medium-sized DRG neurons, or only of TrkA receptors in the case of ND7-23 neurons. A residual neurite growth promoting effect of keratinocytes persisted in a fraction of DRG neurons after Trk receptor blockade. This effect was mimicked by the steroid dehydroepiandrosterone (DHEA) but not by other steroids such as pregnenolone, progesterone or 17β-estradiol. The use of pharmacological agents which inhibit different steps of steroidogenesis indicated that DHEA was probably synthesized from cholesterol in keratinocytes. Our results strongly suggest that DHEA might act as a neurotrophic signal derived from keratinocytes to promote axonal outgrowth from a subpopulation of sensory neurons.     

Distinct requirements for TrkB and TrkC signaling in target innervation by sensory neurons

Signaling by brain-derived neurotrophic factor (BDNF) via the TrkB receptor, or by neurotrophin-3 (NT3) through the TrkC receptor support distinct populations of sensory neurons. The intracellular signaling pathways activated by Trk (tyrosine kinase) receptors, which in vivo promote neuronal survival and target innervation, are not well understood. Using mice with TrkB or TrkC receptors lacking the docking site for Shc adaptors (trkB(shc/shc) and trkC(shc/shc) mice), we show that TrkB and TrkC promote survival of sensory neurons mainly through Shc site-independent pathways, suggesting that these receptors use similar pathways to prevent apoptosis. In contrast, the regulation of target innervation appears different: in trkB(shc/shc) mice neurons lose target innervation, whereas in trkC(shc/shc) mice the surviving TrkC-dependent neurons maintain target innervation and function. Biochemical analysis indicates that phosphorylation at the Shc site positively regulates autophosphorylation of TrkB, but not of TrkC. Our findings show that although TrkB and TrkC signals mediating survival are largely similar, TrkB and TrkC signals required for maintenance of target innervation in vivo are regulated by distinct mechanisms.     

神经损伤对三叉神经中脑核神经元Trk受体表达的影响

本研究应用免疫荧光结合逆行束路追踪技术观察了轴突切断对支配咬肌的三叉神经中脑核(Me5)神经元所含Trk受体蛋白,即TrkA、TrkB和TrkC表达的影响。通过大鼠咬肌神经给予荧光金,标记支配咬肌的Me5神经元;分别于切断咬肌神经后7和14 d对脑切片进行免疫组织化学染色并观察荧光金(FG)标记的Me5神经元表达的三种Trk受体。以双标记神经元占荧光金标记神经元总数的百分率为指标进行统计学分析,结果显示:(1)神经切断后7和14 d,TrkA免疫反应阳性神经元比例有显著性增加(P<0.05);(2)神经切断后随着存活时间的延长,TrkB免疫阳性神经元比例逐渐增加,但无显著性区别(P>0.05);(3)TrkC表达无显著性变化(P>0.05)。本研究结果提示,咬肌神经切断对三种Trk受体的表达有不同的影响,Trk受体表达的模式可能反应出Me5神经元对外周神经损伤后的一种适应。     

Pacinian corpuscle development involves multiple Trk signaling pathways.

The development of crural Pacinian corpuscles was explored in neonatal mutant mice lacking nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) or neurotrophin-4 (NT4), or their cognate Trk receptors. Deficits of the corpuscles and their afferents were greatest in NT3, less in BDNF, and least in NT4 null mice. Deletion of NGF or p75(NTR) genes had little or no impact. No Pacinian corpuscles were present in NT3;BDNF and NT3;NT4 double or NT3;BDNF;NT4 triple null mice. Deficits were larger in NT3 than TrkC mutants and were comparable to deficits observed in TrkB or TrkA mutants. Afferents of all corpuscles coexpressed TrkA and TrkB receptors, and some afferents coexpressed all three Trk receptors. Our results suggest that multiple neurotrophins, in particular NT3, regulate the density of crural Pacinian corpuscles, most likely by regulating the survival of sensory neurons. In addition, NT3/TrkB and/or NT3/TrkA signaling plays a greater role than NT3/TrkC signaling in afferents to developing Pacinian corpuscles.     

Co‐localization of Trk neurotrophin receptors and regulatory peptides in the endocrine cells of the teleostean stomach

Recently it has been observed that a subpopulation of gut endocrine cells in vertebrates express Trk‐like proteins, suggesting that neurotrophins could regulate the synthesis and storage of amines and peptides of these cells. Nevertheless, the peptides and amines present in the endocrine cells that express Trks have not been characterized. In this study we used immunohistochemistry to investigate the occurrence of Trk‐like proteins (TrkA‐like, TrkB‐like and TrkC‐like) and the possible co‐localization of these with peptides and/or biogenic amines in the endocrine cells of the stomach of three teleost (bass, gilt‐head and scorpionfish). No TrkA‐like immunoreactivity (IR) was detected in the stomach of these species, whereas TrkB‐like IR and TrkC‐like IR were observed in numerous cells of the gastric epithelium. TrkB‐like immunoreactive cells were present in all three species examined, and were particularly abundant in the blind sac. Conversely, TrkC‐like immunoreactive cells were found only in the bass stomach, apparently co‐localized with TrkB‐like IR. TrkB‐like IR was found co‐localized with somatostatin IR in scorpionfish, and with somatostatin and CGRP IR in gilt‐head and bass. Gastric endocrine cells expressing 5‐HT, glucagon, insulin, met‐, leu‐enkephalin, substance P, PYY, VIP, CCK, NPY, bombesin and motilin were unreactive for Trk‐like proteins. The present results provide direct evidence for the occurrence of Trk‐like neurotrophin receptor proteins in a subpopulation of the teleostean gastric endocrine cells and suggest that neurotrophins could regulate, as in neurons, the expression of some neuropeptides such as somatostatin and CGRP. Anat Rec 256:219–226, 1999. © 1999 Wiley‐Liss, Inc.     

大鼠Scarpa神经节中Trk受体的免疫组织化学研究

采用免疫组织化学的卵白素-生物素-HRP复合物(ABC)法,以二氨基联苯胺(DAB)为呈色剂,观察高亲和性的神经营养物质受体(酪氨酸激酶受体),即TrkA, TrkB和TrkC在大鼠Scapa神经节(前庭神经节,VG)中的分布。结果显示,许多VG神经元分别对3种Trk受体呈阳性反应,受体位于神经元胞体。每一种Trk受体的反应强度在不同神经元间有区别,有弱、中等和强反应之分。对同一种Trk受体而言,阳性神经元的大小不等;统计结果显示:TrkA, TrkB和TrkC阳性神经元平均面积分别为330.8±7.6, 303.89±10.6和355.05±8.3μm2。该研究结果为Trk受体在VG内发挥维持神经元存活、保持其形态特征等的作用提供了形态学支持。     

The teleost kidney expresses Trk Neurotrophin Receptor-Like Proteins

Neurotrophins are involved in the development and maintenance of some neuronal populations, acting through tyrosine protein kinase (Trk) receptors, TrkA, TrkB and TrkC. In addition, recent studies have demonstrated the occurrence of Trk receptors in a wide variety of adult and embryonic non-neuronal tissues in vertebrates, including kidney. Nevertheless no data are available on Trk proteins distribution in teleost kidney. The present study, by using an immunoperoxidase technique, analyses Trk receptor protein distribution in the kidney of three species of freshwater and saltwater teleost. TrkA-like immunoreactivity was the only one detected in all examined species, while TrkB-like immunoreactivity was completely absent and TrkC-like immunoreactivity was detected only in goldfish. The TrkA immunoreactive cells were mainly localised in the collecting duct system, as this system was the only one to also display TrkC. Such data could provide new clues to Trk in fish and aid assessment of the role of Trk protein receptors during vertebrate evolution. Accepted: 3 November 1999     

The teleost kidney expresses Trk neurotrophin receptor-like proteins

Neurotrophins are involved in the development and maintenance of some neuronal populations, acting through tyrosine protein kinase (Trk) receptors, TrkA, TrkB and TrkC. In addition, recent studies have demonstrated the occurrence of Trk receptors in a wide variety of adult and embryonic non-neuronal tissues in vertebrates, including kidney. Nevertheless no data are available on Trk proteins distribution in teleost kidney. The present study, by using an immunoperoxidase technique, analyses Trk receptor protein distribution in the kidney of three species of freshwater and saltwater teleost. TrkA-like immunoreactivity was the only one detected in all examined species, while TrkB-like immunoreactivity was completely absent and TrkC-like immunoreactivity was detected only in goldfish. The TrkA immunoreactive cells were mainly localised in the collecting duct system, as this system was the only one to also display TrkC. Such data could provide new clues to Trk in fish and aid assessment of the role of Trk protein receptors during vertebrate evolution.     

Association of neurotrophin receptor expression and differentiation in human neuroblastoma.

Interactions of the trk family of tyrosine kinase receptors with neurotrophins result in growth and maturational changes in neuronal cells. The continued progression, maturation, or regression of neuroblastoma, an embryonal, sympathetic nervous system-derived tumor of infants and children, might be governed by neurotrophic influences. Immunocytochemistry was utilized to evaluate TrkA, TrkB, and TrkC protein expression at the cellular level in the developing human fetal sympathetic nervous system and in a selection of neuroblastoma tumor specimens. TrkA and TrkC expression was identified in sympathetic ganglia and within the adrenal medulla, with intense TrkB expression restricted to paraganglia, of the normal developing human sympathetic nervous system. In neuroblastoma, pp140trkA expression correlated positively with favorable tumor stage (P = 0.0027) and favorable outcome (P = 0.026). No statistically significant correlation of TrkC expression with outcome was evident; however, both TrkA and TrkC expression was most apparent in tumor cells of increased differentiation. TrkB expression was primarily localized to cells within the fibrovascular tumor stroma. A model of neurotrophin receptor expression and neurotrophin reactivity with differentiation is proposed. The existence and spatial distribution of neurotrophin receptors in neuroblastoma lend supportive evidence that neurotrophic influences may be involved in tumor persistence or regression.     

Neurotrophin and Trk neurotrophin receptors in the inner ear of Salmo salar and Salmo trutta

Neurotrophins (NTs) and their signal transducing Trk receptors play a critical role in the development and maintenance of specific neuronal populations in the nervous system of higher vertebrates. They are responsible for the innervation of the inner ear cochlear and vestibular sensory epithelia. Neurotrophins and Trks are also present in teleosts but their distribution in the inner ear is unknown. Thus, in the present study, we used Western-blot analysis and immunohistochemistry to investigate the expression and cell localization of both NTs and Trk receptors in the inner ear of alevins of Salmo salar and Salmo trutta. Western-blot analysis revealed the occurrence of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), but not nerve growth factor (NGF), as well as all three Trk receptors, i.e. TrkA, TrkB and TrkC, the estimated molecular weights of which were similar to those expected for mammals. Specific immunoreactivity for neurotrophins was detected mainly in the sensory epithelia. In particular, BDNF immunoreactivity was found in the maculae of the utricle and saccule, whereas NT-3 immunoreactivity was present in the sensory epithelium of the cristae ampullaris. As a rule the sensory epithelia of the inner ear lacked immunoreactivity for Trks, thus excluding possible mechanisms of autocrinia and/or paracrinia. By contrast, overlapping subpopulations of neurons in the statoacoustic ganglion expressed TrkA (about 15%), TrkB (about 65%) and TrkC (about 45%). The present results demonstrate that, as in mammals and birds, the inner ear of teleosts expresses the components of the neurotrophin-Trk system, but their roles remain to be elucidated.     

Neurotrophins and neurotrophin receptors in human lung cancer.

The expression of neurotrophins (NTs) and related high- and low-affinity receptors was studied in surgical samples of histologically diagnosed human tumors of the lower respiratory tract. The experiment was conducted with 30 non-small cell lung cancer specimens and in eight small cell lung cancer specimens by Western blot analysis and immunohistochemistry to assess expression and distribution of NT and NT receptor proteins in tissues examined. Immunoblots of homogenates from human tumors displayed binding of anti-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and NT-3 antibodies as well as of anti-tyrosine-specific protein kinase (Trk) A, TrkB, and TrkC receptor antibodies, with similar migration characteristics than those displayed by human beta-NGF and proteins from rat brain. A specific immunoreactivity for NTs and NT receptors was demonstrated in vessel walls, stromal fibroblasts, immune cells, and sometimes within neoplastic cell bodies. Approximately 33% of bronchioloalveolar carcinomas exhibited a strong membrane NGF and TrkA immunoreactivity, whereas 46% adenocarcinomas expressed an intense TrkA immunoreactivity but a weak immunostaining for NGF within tumor cells. Moreover, squamous cell carcinomas developed an intense TrkA immunoreactivity only within stroma surrounding neoplastic cells. A faint BDNF and TrkB immunoreactivity was documented in adenocarcinomas, squamous cell carcinomas, and small cell lung cancers. NT-3 and its corresponding TrkC receptor were found in a small number of squamous cell carcinomas within large-size tumor cells. No expression of low-affinity p75 receptor protein was found in tumor cells. The detection of NTs and NT receptor proteins in tumors of the lower respiratory tract suggests that NTs may be involved in controlling growth and differentiation of human lung cancer and/or influencing tumor behavior.     

Neurotrophin receptors expression in the developing mouse retina: an immunohistochemical study

 Neurotrophins and their receptors (p75 and Trk family of receptors) play an important role in the survival of different populations of neurons in the central and peripheral nervous system. Expression of p75, TrkA, TrkB and TrkC was examined in mouse retinas by means immunohistochemistry in the postnatal development of normal and rd/rd mice (C57BL/6J). The rd/rd mice suffer a degeneration that causes a massive lost of photoreceptor cells. Results showed immunoreactivity to all three Trk proteins in both normal and rd/rd mice during the first 21 postnatal days, but some variations in intensity and localization were found. p75 immunoreaction was only present in rd/rd mice at the end of the degeneration process. These results could indicate a role of neurotrophins and their receptors in both the postnatal development of mouse retina and the degeneration process of rd/rd mice. Accepted: 30 October 1996     

Differential gene expression pattern of neurotrophins and their receptors during neuronal differentiation of rat bone marrow stromal cells

Neural-like cells derived from bone marrow stromal cells (BMSC) have potential usefulness in repair of the CNS injuries or diseases. The functional recovery mediated by these cells, however, depends on secretion of specific growth factors and their designated receptors. In the present study, we have investigated the expression profile of neurotrophins NGF, BDNF and NT-3 and their high-affinity (TrkA, TrkB, TrkC) and common low-affinity (p75NTR) receptors before and during neural differentiation of rat BMSCs by RT-PCR. Results indicate that NGF and BDNF but not NT-3 are expressed in both un-differentiated as well as neurally differentiated BMSCs. In contrast, the expression of TrkA and TrkB is restricted to neurally differentiated cells, while TrkC is not expressed in these cells either before or after differentiation. Interestingly, p75NTR expression is absent in un-differentiated cells but is initiated upon the induction of neural differentiation, and then shut off in fully differentiated neuron-like cells.     

Nicotine increases the expression of neurotrophin receptor tyrosine kinase receptor A in basal forebrain cholinergic neurons

In this study, we investigated whether the potential positive effects of nicotine in Alzheimer's disease (AD) may involve neurotrophic factors, such as nerve growth factor (NGF), closely associated with basal forebrain (BF) cholinergic function and survival. To this aim, we studied the effects of prolonged nicotine treatment on neurotrophin receptors expression and on NGF protein levels in the rat BF cholinergic circuitry. Both in vivo and in vitro experiments were conducted. We found that s.c. nicotine infusion (1.2 mg free base/kg/d delivered by mini-pumps for 7 days) induced in vivo an increase in tyrosine kinase receptor A (TrkA)—but not TrkB, TrkC or low affinity neurotrophin receptor p75 (p75)—expression in BF cholinergic neurons targeting the cerebral cortex. Nicotine did not produce statistically significant long-lasting effects on NGF levels in the cerebral cortex, or in the BF. In vitro experiments performed on primary BF neuronal cultures, showed that 72 h exposure to nicotine increased both TrkA expression, and NGF release in culture medium. Neutralization experiments with an anti-NGF antibody showed that NGF presence was not necessary for nicotine-induced increase of TrkA levels in cultured cholinergic neurons, suggesting that nicotine may act through NGF-independent mechanisms. This study shows that nicotine, independently of its action on NGF levels, may contribute to the restoration of the trophic support to BF cholinergic neurons by increasing TrkA levels.     

Neurotrophin-3 is a target-derived neurotrophic factor for penile erection-inducing neurons

The aim of this study was to determine whether the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 could act as endogenous target-derived trophic factors for erection-inducing, i.e. penis-projecting major pelvic ganglion (MPG) neurons, and/or penile sensory neurons in adult rat. This was accomplished by studying the expression of NT mRNAs in the penis and their cognate receptors in the MPG and dorsal root ganglia (DRGs), and the retrograde axonal transport of radioiodinated NTs injected into the corpora cavernosa. Northern hybridization showed that NGF, BDNF, and NT-3 mRNAs are expressed in the shaft of the penis. In situ hybridization combined with usage of the retrograde tracer Fluoro-Gold showed that TrkC and p75 receptors are expressed in penis-projecting neurons of the MPG whereas the mRNAs for TrkA and TrkB receptors were undetectable. However, all the NT receptor mRNAs were expressed in penile sensory neurons of sacral level 1 (S1) DRG. (125)I-NT-3 injected into the shaft of the penis was retrogradely transported into the MPG and S1 DRG, whereas radioiodinated NGF and BDNF were transported specifically into the S1 DRG, thus confirming the existence of functional NT receptors in these penile neurons. In conclusion, these data suggest that NT-3 may act as a target-derived neurotrophic factor for both erection-inducing and penile sensory neurons, whereas NGF and BDNF may be more important for the sensory innervation of the penis.