Expression of the TrkA or TrkB receptor tyrosine kinase alters the double-strand break (DSB) repair capacity of SY5Y neuroblastoma cells

In the childhood tumor neuroblastoma, high expression of the TrkA neurotrophin receptor is associated with a favorable prognosis and a lack of structural chromosomal changes, whereas TrkB is expressed in aggressive neuroblastomas demonstrating high genomic instability. The ability to repair DNA double-strand breaks (DSBs) is considered a central determinant of chromosomal stability with nonhomologous end joining (NHEJ) being the major pathway of DSB repair in vertebrates. Here, we used the SH-SY5Y human neuroblastoma cell line ectopically expressing either TrkA or TrkB as a model system to analyze the impact of Trk receptor expression on NHEJ-mediated DSB repair. In a cell-free NHEJ assay, SY5Y-TrkA cells displayed a significantly higher efficiency for NHEJ compared to SY5Y-TrkB cells. To detect possible underlying mechanisms, gene expression data (Affymetrix U95A microarray chips) obtained from the same SY5Y-TrkA/TrkB model system were reanalyzed focussing on genes involved in DNA repair. Expression of XRCC4, a central component of NHEJ, was significantly upregulated in SY5Y-TrkA compared to SY5Y-TrkB cells. Expression data were confirmed using real-time PCR and western blotting. Additionally, XRCC4 expression was enhanced in most primary neuroblastomas with high TrkA expression. The TrkA-induced increase in NHEJ activity could be reverted by XRCC4 knock-down, confirming the induction of XRCC4 by TrkA to be essential for the observed phenotype. Our data provide the first evidence for a functional relationship between tyrosine kinase receptor signaling and NHEJ-mediated DSB repair in cancer cells, potentially contributing to their genomic stability.     

Rotary bioreactor culture can discern specific behavior phenotypes in Trk-null and Trk-expressing neuroblastoma cell lines

Neuroblastoma is characterized by biological and genetic heterogeneity that leads to diverse, often unpredictable, clinical behavior. Differential expression of the Trk family of neurotrophin receptors strongly correlates with clinical behavior; TrkA expression is associated with favorable outcome, whereas TrkB with unfavorable outcome. Neuroblastoma cells cultured in a microgravity rotary bioreactor spontaneously aggregate into tumor-like structures, called organoids. We wanted to determine if the clinical heterogeneity of TrkA- or TrkB-expressing neuroblastomas was reflected in aggregation kinetics and organoid morphology. Trk-null SY5Y cells were stably transfected to express either TrkA or TrkB. Short-term aggregation kinetics were determined by counting the number of single (non-aggregated) viable cells in the supernatant over time. Organoids were harvested after 8 d of bioreactor culture, stained, and analyzed morphometrically. SY5Y-TrkA cells aggregated significantly slower than SY5Y and SY5Y-TrkB cells, as quantified by several measures of aggregation. SY5Y and TrkB cell lines formed irregularly shaped organoids, featuring stellate projections. In contrast, TrkA cells formed smooth (non-stellate) organoids. SY5Y organoids were slightly smaller on average, but had significantly larger average perimeter than TrkA or TrkB organoids. TrkA expression alone is sufficient to dramatically alter the behavior of neuroblastoma cells in three-dimensional, in vitro rotary bioreactor culture. This pattern is consistent with both clinical behavior and in vivo tumorigenicity, in that SY5Y-TrkA represents a more differentiated, less aggressive phenotype. The microgravity bioreactor is a useful in vitro tool to rapidly investigate the biological characteristics of neuroblastoma and potentially to assess the effect of cytotoxic as well as biologically targeted drugs.     

p75 reduces TrkB tyrosine autophosphorylation in response to brain-derived neurotrophic factor and neurotrophin 4/5

Neurotrophins mediate their signals through two different receptors: the family of receptor tyrosine kinases, Trks, and the low affinity pan-neurotrophin receptor p75. Trk receptors show more restricted ligand specificity, whereas all neurotrophins are able to bind to p75. One important function of p75 is the enhancement of nerve growth factor signaling via TrkA by increasing TrkA tyrosine autophosphorylation. Here, we have examined the importance of p75 on TrkB- and TrkC-mediated neurotrophin signaling in an MG87 fibroblast cell line stably transfected with either p75 and TrkB or p75 and TrkC, as well as in PC12 cells stably transfected with TrkB. In contrast to TrkA signaling, p75 had a negative effect on TrkB tyrosine autophosphorylation in response to its cognate neurotrophins, brain-derived neurotrophic factor and neurotrophin 4/5. On the other hand, p75 had no effect on TrkB or TrkC activation in neurotrophin 3 treatment. p75 did not effect extracellular signal-regulated kinase 2 tyrosine phosphorylation in response to brain-derived neurotrophic factor, neurotrophin 3, or neurotrophin 4/5. These results suggest that the observed reduction in TrkB tyrosine autophosphorylation caused by p75 does not influence Ras/mitogen-activated protein kinase signaling pathway in neurotrophin treatments.     

Dok5 is substrate of TrkB and TrkC receptors and involved in neurotrophin induced MAPK activation

Tropomyosin-related kinase (Trk) family receptors are a group of high affinity receptors for neurotrophin growth factors, which have pivotal functions in many physiological processes of nervous system. Trk receptors can dimerize and autophosphorylate upon neurotrophin stimulation, then recruit multiple adaptor proteins to transduct signal. In this report, we identified Dok5, a member of Dok family, as a new substrate of TrkB/C receptors. In yeast two-hybrid assay, Dok5 can interact with intracellular domain of TrkB and TrkC receptor through its PTB domain, but not with that of TrkA receptor. The interaction was then confirmed by GST pull-down assay and Co-IP experiment. Dok5 co-localized with TrkB and TrkC in differentiated PC12 cells, providing another evidence for their interaction. By using mutational analysis, we characterized that Dok5 PTB domain bound to Trk receptor NPQY motif in a kinase-activity-dependent manner. Furthermore, competition experiment indicated that Dok5 competed with N-shc for binding to the receptors at the same site. Finally, we showed that Dok5 was involved in the activation of MAPK pathway induced by neurotrophin stimulation. Taken together, these results suggest that Dok5 acts as substrate of TrkB/C receptors and is involved in neurotrophin induced MAPK signal pathway activation.     

A novel endocytic recycling signal distinguishes biological responses of Trk neurotrophin receptors

Endocytic trafficking of signaling receptors to alternate intracellular pathways has been shown to lead to diverse biological consequences. In this study, we report that two neurotrophin receptors (tropomyosin-related kinase TrkA and TrkB) traverse divergent endocytic pathways after binding to their respective ligands (nerve growth factor and brain-derived neurotrophic factor). We provide evidence that TrkA receptors in neurosecretory cells and neurons predominantly recycle back to the cell surface in a ligand-dependent manner. We have identified a specific sequence in the TrkA juxtamembrane region, which is distinct from that in TrkB receptors, and is both necessary and sufficient for rapid recycling of internalized receptors. Conversely, TrkB receptors are predominantly sorted to the degradative pathway. Transplantation of the TrkA recycling sequence into TrkB receptors reroutes the TrkB receptor to the recycling pathway. Finally, we link these divergent trafficking pathways to alternate biological responses. On prolonged neurotrophin treatment, TrkA receptors produce prolonged activation of phosphatidylinositol 3-kinase/Akt signaling as well as survival responses, compared with TrkB receptors. These results indicate that TrkA receptors, which predominantly recycle in signal-dependent manner, have unique biological properties dictated by its specific endocytic trafficking itinerary.     

p75 neurotrophin receptor reduces ligand-induced Trk receptor ubiquitination and delays Trk receptor internalization and degradation

Target-derived neurotrophins regulate neuronal survival and growth by interacting with cell-surface tyrosine kinase receptors. The p75 neurotrophin receptor (p75 NTR) is coexpressed with Trk receptors in long-range projection neurons, in which it facilitates neurotrophin binding to Trk and enhances Trk activity. Here, we show that TrkA and TrkB receptors undergo robust ligand-dependent ubiquitination that is dependent on activation of the endogenous Trk activity of the receptors. Coexpression of p75 NTR attenuated ubiquitination of TrkA and TrkB and delayed nerve growth factor-induced TrkA receptor internalization and receptor degradation. These results indicate that p75 NTR may prolong cell-surface Trk-dependent signalling events by negatively regulating receptor ubiquitination.     

ShcB and ShcC activation by the Trk family of receptor tyrosine kinases

Activation of the neurotrophin Trk receptors is a key process in the survival and development of the nervous system. The signaling adapters ShcB and ShcC, but not ShcA, are thought to be the primary Shc adaptor proteins in neurons as both are highly expressed in both the developing and adult nervous system. Although a previous study suggested that ShcB and ShcC do not strongly interact with the Trk receptors (1), we find that ShcB and ShcC bind the Trk receptors in a phosphotyrosine-dependent manner via their N-terminal phosphotyrosine binding domain at Tyr(499) (TrkA) and Tyr(515) (TrkB), they are tyrosine-phosphorylated in response to neurotrophin stimulation, and they enhance the activation of mitogen-activated protein kinase in Trk-expressing cells. Moreover, neurotrophin treatment of primary cortical neurons stimulates ShcB/ShcC-Trk interaction and the tyrosine phosphorylation of ShcB/ShcC, indicating that they are bona fide targets of the Trk receptors in vivo. Interestingly, two proteins (pp60 and pp75) co-immunoprecipitate with ShcB and ShcC in response to neurotrophin stimulation in primary cortical neurons, suggesting a potential role of these unknown targets in neurotrophin signaling. Collectively, these results demonstrate that ShcB and ShcC, and their co-immunoprecipitating proteins, are activated by the Trk receptors in primary neurons.     

The Crystal Structures of TrkA and TrkB Suggest Key Regions for Achieving Selective Inhibition

The Trk family of neurotrophin receptors, which includes the three highly homologous proteins TrkA, TrkB and TrkC, is strongly associated with central and peripheral nervous system processes. Trk proteins are also of interest in oncology, since Trk activation has been observed in several cancer types. While Trk kinases are attractive oncology targets, selectivity might be more of an issue than for other kinases due to potential CNS side effects if several Trk kinases are simultaneously targeted. In order to address this issue, we present here the first structures of human TrkA and TrkB kinase domains and three complexes between TrkB and Trk inhibitors. These structures reveal different conformations of the kinase domain and suggest new regions of selectivity among the Trk family.     

Generation of TrkA/TrkB Chimeric Receptor Constructs Reveals Molecular Mechanisms Underlying BDNF-Induced Dendritic Outgrowth in Hippocampal Neurons

Neurotrophins (NTs) regulate neuronal survival, differentiation, and synaptic plasticity through tropomyosin receptor kinases (Trks). The molecular mechanisms underlying these functions, however, have remained incompletely understood. In the present study, we first showed that brain-derived neurotrophic factor (BDNF) increased both the number of primary dendrites and dendritic complexity in cultured hippocampal neurons. Since hippocampal neurons predominantly express the BDNF receptor TrkB, but not the nerve growth factor (NGF) receptor Trk, we generated DNA constructs encoding the extracellular domain of TrkA fused with the transmembrane and intracellular domain of TrkB and introduced these constructs into cultured hippocampal neurons. To visualize the dendrites, the TrkA/TrkB fusion proteins were bicistronically expressed with green fluorescence protein (GFP). Interestingly, the GFP-labeled neurons grew dendrites and activated the TrkA/TrkB receptors in response to NGF, but not BDNF. We next generated a series of TrkA/TrkB receptors with mutations at tyrosine residues in the TrkB kinase domain, and sought to identify the signaling pathway required for NT-induced dendrite outgrowth. Sholl analyses demonstrated that TrkB signaling through Shc, but not through PLC-γ, plays a crucial role in NT-elicited dendritic outgrowth in hippocampal neurons.     

TrkA induces apoptosis of neuroblastoma cells and does so via a p53-dependent mechanism

Neuroblastoma (NB) is the most frequent solid extracranial tumor in children. Its clinical prognosis correlates with the expression of members of the Trk neurotrophin receptor family, which includes TrkA and TrkB. TrkA expression is associated with favorable prognosis, whereas TrkB expression is associated with poor prognosis. Here we show that TrkA expression induces the apoptosis of NB cells and does so by modulating the levels or activities of a number of proteins involved in regulating cell survival and apoptosis, including p53, Bcl-2, and caspase-3. TrkA increased the expression of p53 target proteins and failed to induce apoptosis in cells where p53 was inactivated by mutation or via expression of dominant inhibitory p53 or E1B55K, indicating that TrkA mediates apoptosis, at least in part, through p53. Treatment with a caspase inhibitor or overexpression of Bcl-X(L) also prevented TrkA from inducing apoptosis. In contrast, elevated expression of TrkA in non-transformed sympathetic neurons resulted in the suppression of p53 levels and enhanced survival. These results identify apoptosis as a novel biological response of TrkA in NB cells and imply that TrkA is a good prognosis marker for NB due in part to its ability to mediate apoptosis when expressed at sufficient levels.     

Effects of castration on the immunoreactivity to NGF, BDNF and their receptors in the pelvic ganglia of the male rat

Nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and are members of the neurotrophin family, a family of neurotrophic factors that also includes neurotrophin (NT) 3 and NT4/5. Neurotrophins have essential roles in the survival, development and differentiation of neurons in the central and peripheral nervous systems. Neurotrophins exert their effects by binding to corresponding receptors which are formed by the tyrosine protein kinases TrkA, TrkB and TrkC, and the low affinity neurotrophic receptor (p75NTR). In the present study, using immunohistochemistry and quantitative analysis, we have investigated immunoreactivity to BDNF, NGF, TrkB, p75NTR and TrkA in the pelvic ganglia of normal and castrated rats. Neurons of the pelvic ganglia expressed both these neurotrophins and their receptors. After castration the immunoreactivity persisted. However, the number of BDNF- and p75NTR-IR cells statistically significant decreased after castration. These results suggest that castration modulates the expression of neurotrophins and their receptors in pelvic autonomic neurons.     

Cell survival through Trk neurotrophin receptors is differentially regulated by ubiquitination

Specificity of neurotrophin factor signaling is dictated through the action of Trk receptor tyrosine kinases. Once activated, Trk receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin receptors are differentially regulated by ubiquitination to modulate the survival of neurons.     

Proneurotrophins require endocytosis and intracellular proteolysis to induce TrkA activation

The uncleaved, pro-form of nerve growth factor (proNGF) functions as a pro-apoptotic ligand for the p75 neurotrophin receptor (p75NTR). However, some reports have indicated that proneurotrophins bind and activate Trk receptors. In this study, we have examined proneurotrophin receptor binding and activation properties in an attempt to reconcile these findings. We show that proNGF readily binds p75NTR expressed in HEK293T cells but does not interact with TrkA expressed under similar circumstances. Importantly, proNGF activates TrkA tyrosine phosphorylation, induces Erk and Akt activation, and causes PC12 cell differentiation. We show that inhibiting endocytosis or furin activity reduced TrkA activation induced by proNGF but not that induced by mature NGF and that proNGF123, a mutant form of NGF lacking dibasic cleavage sites in the prodomain, does not induce TrkA phosphorylation in PC12 cells. Therefore, endocytosis and cleavage appear to be prerequisites for proNGF-induced TrkA activity. We also found that proBDNF induces activation of TrkB in cerebellar granule neurons and that proBDNF cleavage by furin and metalloproteases facilitates this effect. Taken together, these data indicate that under physiological conditions, proneurotrophins do not directly bind or activate Trk receptors. However, endocytosis and cleavage of proneurotrophins produce processed forms of neurotrophins that are capable of inducing Trk activation.     

大鼠脑内小胶质细胞神经营养素受体的表达

神经营养素在神经元的生长、发育中的重要作用已有许多报道,但对神经胶质细胞的作用及其作用机制却知之甚少。在本研究中,我们着重对体外培养的小胶质细胞所表达的神经营养素受体进行了分析。首先,利用酚-氯仿法提取了总的细胞RNA,然后通过特异引物采用反转录多聚酶链式反应(RT-PCR)扩增得到cDNA,再用琼脂糖凝胶电泳、DNA印迹法和免疫细胞化学染色法对神经营养素受体(Trks)进行了测定。实验结果表明:体外培养的大鼠脑小胶质细胞表达高亲和力神经营养素受体TrkA、TrkB和TrkC,以及低亲和力NGF受体LNGFRp75。因此推断,神经营养素对小胶质细胞的生理及调节作用可能是通过它们相应的受体(Trks和LNGFRp75)介导的。这些结果为进一步研究神经营养素在神经系统中的作用机制及小胶质细胞的生理功能提供了资料。     

Brain ischemia, neurogenesis, and neurotrophic receptor expression in primates

Generation of new neurons persists in the normal adult mammalian brain, with neural stem/progenitor cells residing in at least two brain regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Adult neurogenesis is well documented in the rodent, and has also been demonstrated in vivo in nonhuman primates and humans. Brain injuries such as ischemia affect neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in SGZ or SVZ. We addressed the issue whether an injury triggered activation of endogenous neuronal precursors also takes place in the adult primate brain. We found that the ischemic insult increased the number of progenitor cells in monkey SGZ and SVZ, and caused gliogenesis in the ischemia-prone hippocampal CA1 sector. To better understand the mechanisms regulating precursor cell division and differentiation in the primate, we analyzed the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors. We found that a fraction of mitotic progenitors were positive for the neurotrophin receptor TrkB, while immature neurons expressed the neurotrophin receptor TrkA. Astroglia, ependymal cells and blood vessels in SVZ were positive for distinctive sets of ligands/receptors, which we characterized. Thus, a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate SVZ. We also analyzed microglial and astroglial proliferation in postischemic hippocampal CA1 sector. We found that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and the tyrosine kinase TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus.     

Trypanosome trans-sialidase mediates neuroprotection against oxidative stress, serum/glucose deprivation, and hypoxia-induced neurite retraction in Trk-expressing PC12 cells

Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme and a novel ligand of tyrosine kinase (TrkA) receptors but not of neurotrophin receptor p75NTR. Here, we show that TS targets TrkB receptors on TrkB-expressing pheochromocytoma PC12 cells and colocalizes with TrkB receptor internalization and phosphorylation (pTrkB). Wild-type TS but not the catalytically inactive mutant TSDeltaAsp98-Glu induces pTrkB and mediates cell survival responses against death caused by oxidative stress in TrkA- and TrkB-expressing cells like those seen with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). These same effects are not observed in Trk deficient PC12(nnr5) cells, but are re-established in PC12(nnr5) cells stably transfected with TrkA or TrkB, are partially blocked by inhibitors of tyrosine kinase (K-252a), mitogen-activated protein/mitogen-activated kinase (PD98059) and completely blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). Both TrkA- and TrkB-expressing cells pretreated with TS or their natural ligands are protected against cell death caused by serum/glucose deprivation or from hypoxia-induced neurite retraction. The cell survival effects of NGF and BDNF against oxidative stress are significantly inhibited by the neuraminidase inhibitor, Tamiflu. Together, these observations suggest that trypanosome TS mimics neurotrophic factors in cell survival responses against oxidative stress, hypoxia-induced neurite retraction and serum/glucose deprivation.     

Neurotrophin receptor homolog-2 regulates nerve growth factor signaling

The neurotrophin receptor homolog (NRH2) is closely related to the p75 neurotrophin receptor (p75NTR); however, its function and role in neurotrophin signaling are unclear. NRH2 does not bind to nerve growth factor (NGF), however, is able to form a receptor complex with tropomyosin-related kinase receptor A (TrkA) and to generate high-affinity NGF binding sites. Despite this, the mechanisms underpinning the interaction between NRH2 and TrkA remain unknown. Here, we identify that the intracellular domain of NRH2 is required to form an association with TrkA. Our data suggest extensive intracellular interaction between NRH2 and TrkA, as either the juxtamembrane or death domain regions of NRH2 are sufficient for interaction with TrkA. In addition, we demonstrate that TrkA signaling is dramatically influenced by the co-expression of NRH2. Importantly, NRH2 did not influence all downstream TrkA signaling pathways, but rather exerted a specific effect, enhancing src homology 2 domain-containing transforming protein (Shc) activation. Moreover, downstream of Shc, the co-expression of NRH2 resulted in TrkA specifically modulating mitogen-activated protein kinase pathway activation, but not the phosphatidylinositol 3-kinase/Akt pathway. These results indicate that NRH2 utilizes intracellular mechanisms to not only regulate NGF binding to TrkA, but also specifically modulate TrkA receptor signaling, thus adding further layers of complexity and specificity to neurotrophin signaling.     

Induction of a Distinct Morphology and Signal Transduction in TrkB/PC12 Cells by Nerve Growth Factor and Brain-Derived Neurotrophic Factor

Abstract: A clonal cell line stably expressing trkB (TrkB/PC12) was established from rat pheochromocytoma PC12 cells. Brain-derived neurotrophic factor (BDNF), as well as nerve growth factor (NGF), stimulates neurite outgrowth in TrkB/PC12 cells. However, the morphology of BDNF-differentiated cells was clearly different from NGF-differentiated cells. BDNF treatment brought about longer and thicker neurites and induced a flattened soma and an increase in somatic size. This is not explained enough by the quantitative difference in the strength between TrkA and TrkB stimulation, because the level of BDNF-stimulated tyrosine phosphorylation of TrkB was similar to that of TrkA stimulated with NGF in PC12/TrkB cells. There was no difference in major tyrosine phosphorylated proteins induced by NGF and BDNF. Signal proteins such as phosphatidylinositol 3-kinase, phospholipase C-γ1, Shc, and mitogen-activated protein kinase seem to be involved in both TrkA- and TrkB-mediated signaling pathways. However, a tyrosine-phosphorylated 38-kDa protein (pp38) was detected in anti-pan-Trk immunoprecipitation only after NGF stimulation. Immunoprecipitation using three distinct anti-pan-Trk antibodies suggests that pp38 is not a fragment of TrkA. These data indicate that TrkA has a unique signal transduction pathway that is not stimulated through TrkB in TrkB/PC12 cells and suggest distinct functions among neurotrophin receptors.