NTRK3 overexpression in undifferentiated sarcomas with YWHAE and BCOR genetic alterations.

The BCOR family of tumors includes a number of undifferentiated sarcomas, occurring in various age groups and anatomic sites, characterized by a spindle and round cell phenotype and diffuse immunoreactivity for BCOR. Prior RNA sequencing data revealed that NTRK3 was a top-upregulated gene in BCOR-CCNB3 sarcomas. In this study, we investigate a large cohort of tumors harboring BCOR/YWHAE genetic alterations for NTRK3 upregulation at both the mRNA and protein levels, compared with other sarcoma types. Pan-Trk immunohistochemistry was assessed for intensity and extent. A correlation between NTRK3 expression and the type of BCOR alteration and BCOR immunoreactivity was also performed. Most soft tissue undifferentiated round cell sarcomas with YWHAE or BCOR rearrangements or BCOR internal tandem duplications (ITD) showed NTRK3, but not NTRK1 or NTRK2, upregulation by RNA sequencing data analysis. Cytoplasmic pan-Trk immunoreactivity was also observed in most soft tissue round cell sarcomas with YWHAE rearrangements (100%), BCOR ITD (80%), and BCOR-CCNB3 fusions (67%), as well as clear cell sarcomas of kidney (75%), another BCOR family tumor, and ossifying fibromyxoid tumors with ZC3H7B-BCOR fusion (100%), with variable staining intensity and extent. Pan-Trk staining was also seen in solitary fibrous tumors (100%) and less frequently in synovial sarcoma and Ewing sarcoma, but rarely in other sarcomas tested. Tumors harboring rare fusion variants of BCOR, such as BCOR-CHD9, a novel fusion identified by targeted RNA sequencing, and KMT2D-BCOR, were also positive for pan-Trk staining and NTRK3 overexpression. In conclusion, NTRK3 upregulation resulting in pan-Trk overexpression is common in the BCOR family of tumors as well as in subsets of BCOR-expressing sarcomas through alternative mechanisms. The therapeutic implication of this finding awaits further investigation.

Exogenous Expression of Nt-3 and TrkC Genes in Bone Marrow Stromal Cells Elevated the Survival Rate of the Cells in the Course of Neural Differentiation.

Bone marrow stromal cells (BMSCs) are attractive cellular sources for cell therapy of many diseases, specifically neurodegenerative ones. The potential capability of BMSCs could be further augmented by enhancing their neuroprotective property, differentiation potential, and survival rate subsequent to transplantation. Therefore, a concurrent upregulation of neurotrophin-3 (NT-3) and its high affinity receptor, tyrosin kinase C (TrkC), was utilized in our study. BMSCs were cotransfected with pDsRed1-N1-NT-3 and pCMX-TrkC plasmids before induction of neural differentiation. pEGFP-N1-transfected BMSCs were also employed as a control. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed for gene expression analysis. Cell viability was evaluated by MTT assay, while apoptosis rate was assessed by flow cytometry after PI and Annexin V staining. NT-3 and TrkC mRNA levels were greatly elevated following cotransfection of cells with pDsRed1-N1-NT-3 and pCMX-TrkC vectors. The expression of neural markers (i.e., NFM, and NeuroD1) was augmented in cotransfected BMSCs, compared to the control ones, after neural induction. At each time point, the viability and apoptosis rates of the cells over-expressing NT-3 and TrkC showed increased and reduced patterns, respectively. Our data demonstrated that NT-3/TrkC-co-transfected BMSCs, compared to those of intact cells, could be more beneficial graft candidates for the upcoming treatment strategies of neurogenic disorders due to their increased viability and expression of neural markers. This may be due to their increased level of neural differentiation potential and/or their enhanced rate of survival and/or their useful capacity to secrete NT-3.

Tropomyosin receptor kinase C (TrkC) expression in medulloblastoma: relation to the molecular subgroups and impact on treatment response.

PURPOSE: High messenger RNA (mRNA) expression of the tropomyosin receptor kinase C gene (TrkC) has been associated with favorable survival in medulloblastoma patients. Untested is whether it plays a role through modulating the response to therapy or whether it might be a surrogate marker for a favorable molecular subgroup. METHODS: The medulloblastoma-derived cell line DAOY was stably transfected to overexpress TrkC (clone DAOY-TrkC) and compared to a control (clone DAOY-EV, empty vector transfected). Cell viability (MTS assay) was tested after irradiation or incubation with chemotherapeutic drugs. Neuroradiologic response to postoperative chemotherapy or craniospinal irradiation (CSI) of medulloblastoma patients aged 3-21 years with postoperative residual disease treated within the consecutive trials HIT'91/HIT2000 was compared to TrkC mRNA expression in their tumor samples. Five well-characterized independent expression-profiling studies covering together 686 medulloblastoma patients were analyzed for TrkC levels according to the molecular subgroups. RESULTS: Cell viability of DAOY-TrkC compared to DAOY-EV was not different after exposure to increasing doses of irradiation, cisplatin, etoposide, or vincristine. While TrkC mRNA expression tended to be higher in non-responders (n = 5/19) to postoperative CSI (p = 0.03, ratio 15.5, 95% CI 9-267), this was the case in responders (n = 23/43) to chemotherapy (p = 0.04, ratio 6.1, 95% CI 1.1-35), both analyzed with Mann-Whitney U test (not significant after Bonferroni adjustment). The highest TrkC mRNA levels were found in the SHH subgroup across all expression-profiling studies. CONCLUSIONS: High TrkC mRNA expression appears to be frequent in the SHH subgroup and seems not to have a major effect on therapy responsiveness in medulloblastoma patients.

Developmental changes in the protective effect of exogenous brain-derived neurotrophic factor and neurotrophin-3 against ototoxic drugs in cultured rat vestibular ganglion neurons.

We examine developmental changes in the responsiveness of rat vestibular ganglion neurons (VGNs) to two neurotrophic factors (NTFs), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and investigate the protective effects of these NTFs against ototoxic drugs during postnatal development in dissociated cultures. VGNs were obtained from rats on postnatal days (P) 1, 3, 7 and 14. BDNF facilitated neuronal survival as well as neurite sprouting of VGNs obtained from younger rats (P1 and P3), whereas these effects were not observed in older rats (P7 and P14). BDNF was also effective in facilitating neurite extension in VGNs at each of the postnatal ages. NT-3 also facilitated neuronal survival and neurite extension of VGNs from younger rats but these effects were significantly smaller than those of BDNF (p < 0.05). The protective effects of BDNF and NT-3 against ototoxic drugs, gentamicin and cisplatin, were also age-dependent: they were effective for neuronal survival, neurite sprouting and neurite extension in VGNs from younger rats, whereas these effects tended to disappear in VGNs from older rats. Analysis of the changes in the expression of the receptors of NTFs revealed that expression of TrkB and TrkC proteins and their mRNA did not change during the developmental period, whereas expression of p75(NTR) protein was down-regulated together with that of p75(NTR) mRNA during the developmental period. Developmental changes in the responsiveness to exogenous NTFs in VGNs, which is not caused by the changes of their receptors but probably caused by changes in the intracellular signaling pathways, should be taken into consideration in the prevention of neuronal degeneration caused by ototoxic drugs.

Expression of tyrosine kinase receptors in cultured dorsal root ganglion neurons in the presence of monosialoganglioside and skeletal muscle cells.

The neurotrophic factor-like activity of monosialoganglioside (GM1) has been shown to activate tyrosine kinase receptors (Trk). Targets of neuronal innervation play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. Both GM1 and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons. However, much less is known about the effects of GM1 or/and target SKM cells on the expression of Trk receptors in dorsal root ganglion (DRG) neurons. Here we have tested what extent to the expression of TrkA, TrkB, and TrkC receptors in primary cultured of DRG neurons in absence or presence of GM1 or/and SKM cells. In this experiment, we found that: (1) GM1 promoted expression of TrkA and TrkB but not TrkC in primary cultured DRG neurons; (2) target SKM cells promoted expression of TrkC but not TrkA and TrkB in neuromuscular cocultures without GM1 treatment; and (3) GM1 and target SKM cells had additional effects on expression of these three Trk receptors. The results of the present study offered new clues for a better understanding of the association of GM1 and target SKM on the expression of Trk receptors.

Neurotrophin and GDNF family ligand receptor expression in vagal sensory nerve subtypes innervating the adult guinea pig respiratory tract.

We combined retrograde tracing techniques with single-neuron RT-PCR to compare the expression of neurotrophic factor receptors in nodose vs. jugular vagal sensory neurons. The neurons were further categorized based on location of their terminals (tracheal or lungs) and based on expression of the ionotropic capsaicin receptor TRPV1. Consistent with functional studies, nearly all jugular neurons innervating the trachea and lungs expressed TRPV1. With respect to the neurotrophin receptors, the TRPV1-expressing jugular C-fiber neurons innervating both the trachea and lung compartments preferentially expressed tropomyosin-receptor kinase A (TrkA), with only a minority of neurons expressing TrkB or TrkC. The nodose neurons that express TRPV1 (presumed nodose C-fibers) innervate mainly intrapulmonary structures. These neurons preferentially expressed TrkB, with only a minority expressing TrkA or TrkC. The expression pattern in tracheal TRPV1-negative neurons, nodose tracheal presumed Aδ-fiber neurons as well as the intrapulmonary TRPV1-negative presumed Aß-fiber neurons, was similar to that observed in the nodose C-fiber neurons. We also evaluated the expression of GFRα receptors and RET (receptors for the GDNF family ligands). Virtually all vagal sensory neurons innervating the respiratory tract expressed RET and GFRα1. The jugular neurons also categorically expressed GFRα3, as well as ∼50% of the nodose neurons. GFRα2 was expressed in ∼50% of the neurons irrespective of subtype. The results reveal that Trk receptor expression in vagal afferent neurons innervating the adult respiratory tract depends more on the location of the cell bodies (jugular vs. nodose ganglion) than either the location of the terminals or the functional phenotype of the nerve. The data also reveal that in addition to neurotrophins, the GDNF family ligands may be important neuromodulators of vagal afferent nerves innervating the adult respiratory tract.

Large cell/anaplastic medulloblastoma: outcome according to myc status, histopathological, and clinical risk factors.

PURPOSE: To evaluate the prognostic impact of large cell/anaplastic (LC/A) histology together with molecular and clinical risk factors in childhood medulloblastoma. METHODS: Three consecutive prospective medulloblastoma trials were screened for patients with the histological diagnosis of LC/A medulloblastoma. Tumors were considered as LC/A if they displayed areas of severe cytological anaplasia or a significant or predominant large cell component. Histology was centrally confirmed. Genomic DNA amplification of c-myc and n-myc, and mRNA expression of c-myc and trkC were analyzed. RESULTS: Twenty-eight patients with LC/A medulloblastoma with a median age of 6.1 years (1.4-16.5 years) and a median follow-up of 4.5 years were identified (5% of all medulloblastoma). Four-year event-free (EFS) and overall survival (OS) were 58% and 67%. Young age and metastases (n = 13, 4-year EFS 31% vs. 82% in 15 children >4 years and without metastases, P = 0.001), large cell histology (n = 9, 4-year EFS 22% vs. 75%, P = 0.005) and c-myc amplification (n = 9, 4-year EFS 22% vs. 89%, P < 0.0001) were negative prognostic factors. C-myc amplification was highly correlated with young age (P < 0.001), metastases (P = 0.002) and large cell histology (P = 0.007). Outcome of 12 patients with severely anaplastic tumors without these risk factors was not impaired (4-year EFS 86%). CONCLUSION: In a subgroup of patients without clinical and molecular risk factors outcome was favorable despite severely anaplastic histology. In contrast, c-myc amplification and large-cell histology were associated with an inferior outcome. Intensified treatment strategies should be considered for children with LC/A medulloblastoma and these characteristics.

Trk receptor expression and inhibition in neuroblastomas.

Neuroblastoma, the most common and deadly solid tumor in children, exhibits heterogeneous clinical behavior, from spontaneous regression to relentless progression. Current evidence suggests that the TRK family of neurotrophin receptors plays a critical role in these diverse behaviors. Neuroblastomas expressing TrkA are biologically favorable and prone to spontaneous regression or differentiation, depending on the absence or presence of its ligand (NGF) in the microenvironment. In contrast, TrkB-expressing tumors frequently have MYCN amplification and are very aggressive and often fatal tumors. These tumors also express the TrkB ligand (BDNF), resulting in an autocrine or paracrine survival pathway. Exposure to BDNF promotes survival, drug resistance, and angiogenesis of TrkB-expressing tumors. Here we review the role of Trks in normal development, the different functions of Trk isoforms, and the major Trk signaling pathways. We also review the roles these receptors play in the heterogeneous biological and clinical behavior of neuroblastomas, and the activation of Trk receptors in other cancers. Finally we address the progress that has been made in developing targeted therapy with Trk-selective inhibitors to treat neuroblastomas and other tumors with activated Trk expression.

TrkC binds to the bone morphogenetic protein type II receptor to suppress bone morphogenetic protein signaling.

TrkC, a member of the tropomyosin-related kinase (Trk) family of neurotrophin receptors, is implicated in the growth and survival of human cancer tissues. TrkC is also a potent oncoprotein expressed in tumors derived from multiple cell lineages, and functions as an active protein tyrosine kinase by neurotrophin-3 (NT-3). We previously reported that TrkC plays an essential role in tumor growth and metastasis in a murine cancer cell line. Here, we report that expression of TrkC suppresses bone morphogenetic protein 2 (BMP-2)-induced Smad1 phosphorylation and transcriptional activation. In the highly metastatic CT26 murine colon cancer cell line, which expresses endogenous TrkC, silencing TrkC expression by small interfering RNA significantly enhanced BMP-2-induced Smad1 phosphorylation and restored BMP-2 growth inhibitory activity. In contrast, expression of TrkC in RIE-1 cells, in which TrkC is not expressed, completely suppressed BMP-2 transcriptional activation. Furthermore, we showed that TrkC directly binds to the BMP type II receptor (BMPRII), thereby preventing it from interacting with the BMPRI. This activity requires a functional TrkC protein tyrosine kinase, and the BMPRII seems to be a direct target of TrkC. Our findings provide evidence for a previously unknown mechanism by which TrkC, a neuronal receptor, can block BMP tumor-suppressor activity.

Expression of trkB and trkC receptors and their ligands brain-derived neurotrophic factor and neurotrophin-3 in the murine amygdala.

The neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their cognate receptors, trkB and trkC, have a variety of physiological brain functions, ranging from cell survival to mechanisms involved in learning and memory and long-term potentiation (LTP). LTP can be induced in the cortex and hippocampus, as well as within the amygdala. However, the role of neurotrophins in amygdalar LTP is largely unknown. Expression patterns of BDNF and NT-3 and their cognate receptors in the adult mouse amygdala have not been analyzed in detail. We have therefore examined the expression of trkB, trkC, BDNF, and NT-3 mRNA and protein in different amygdalar nuclei as well as in the hippocampal areas CA1-CA3 and the dentate gyrus. The distribution pattern of trkB, trkC, BDNF, and NT-3 mRNA in the murine hippocampus is comparable to that seen in rats. Within most amygdalar nuclei, a moderate BDNF mRNA expression was found; however, BDNF mRNA was virtually absent from the central nucleus. No expression of NT-3 mRNA was found within the amygdala, but trkC mRNA-expressing cells were widely distributed within this brain region. trkB mRNA was strongly expressed in the amygdala. Because trkB is expressed in a full-length and a truncated form (the latter form is also expressed by nonneuronal cells), we also investigated the distribution of full-length trkB mRNA-expressing cells and could demonstrate that this version of trkB receptors is also widely expressed in the amygdala. These results can serve as a basis for studies elucidating the physiological roles of these receptors in the amygdala.

Neurotrophin receptor expression in human primary retinoblastomas and retinoblastoma cell lines.

BACKGROUND: Neurotrophin receptor signaling regulates proliferation, differentiation and death of neuronal cells. Expression of Trk receptors has been implicated in the pathogenesis and prognosis of embryonal tumors, including neuroblastoma, nephroblastoma, and medulloblastoma. PROCEDURE: We analyzed TrkA, TrkB, TrkC, and p75 expression using semi-quantitative RT-PCR in 23 retinoblastomas and 8 retinoblastoma cell lines. Comparison of mRNA expression with clinical variables as well as the proliferation (PI) and apoptotic index (AI) of the tumor, was performed by Pearson correlation analysis and two-sample t-test. RESULTS: Almost all tumor samples and cell lines demonstrated high expression of all Trk receptors. Expression of TrkB and its ligand, BDNF, was most pronounced, suggesting TrkB to be the major Trk receptor involved in retinoblastoma biology. In contrast, p75 expression was substantially reduced in a subset of tumors and cell lines, in particular compared to its expression in normal retina. Tumors with infiltrative growth demonstrated significantly lower relative levels of TrkC expression than localized tumors (P = 0.004). High expression of TrkA was associated with a higher AI (P = 0.04), and high expression of TrkC was associated with a younger age of the patients (P = 0.03). Inhibition of Trk signaling by K252a resulted in marked growth inhibition of retinoblastoma cells in vitro. CONCLUSIONS: Our findings suggest a role for neurotrophin signaling in the biology of retinoblastoma. General Trk inhibitors are effective in decreasing growth rates of retinoblastoma cells in vitro, and should be evaluated in in vivo studies.

Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate.

Tactile information is perceived by a heterogeneous population of specialized neurons. Neurotrophin receptors (the receptor tyrosine kinases, Trks) mark the major classes of these sensory neurons: TrkA is expressed in neurons that sense temperature and noxious stimuli, and TrkC is expressed in proprioceptive neurons that sense body position. Neurotrophin signaling through these receptors is required for cell survival. To test whether neurotrophins have an instructive role in sensory specification, we expressed rat TrkC from the TrkA (also known as Ntrk1) locus in mice. The surviving presumptive TrkA-expressing neurons adopted a proprioceptive phenotype, indicating that neurotrophin signaling can specify sensory neuron subtypes.

Expression of mRNA of neurotrophic factors and their receptors are significantly altered after subchronic ketamine treatment.

The neurotrophic factors play an important role in the maintenance of neurone viability and neuronal communication which are considered to be altered in schizophrenia. Subchronic application of ketamine (Ket) was found to be a useful model in schizophrenia research. To further validate this model the mRNA levels of neurotrophic factors NGF, NT-3, and BDNF and their receptors TrkA, TrkB, and TrkC, respectively, were measured in different brain areas in Ket-pretreated rats subchronically dosed with the atypical antipsychotic drug risperidone (Ris). With the exception of NGF in the frontal cortex, Ket pretreatment did change NGF, NT-3, and BDNF mRNA levels in the frontal cortex, the hippocampus, the striatum, the thalamus/hypothalamus region, and in the cerebellum. These changes correspond with changes at their tyrosine kinase receptors. Ris treatment normalised altered NT-3 levels in the hippocampus and balanced BDNF levels in the same structure. It was concluded that the Ket model might reflect distinct alterations in neurotrophic factor activity as found in schizophrenic patients and, moreover, that Ris treatment rebalances disturbed neurotrophic factor activity.

Neurotrophin-3 administration alters neurotrophin, neurotrophin receptor and nestin mRNA expression in rat dorsal root ganglia following axotomy.

In the months following transection of adult rat peripheral nerve some sensory neurons undergo apoptosis. Two weeks after sciatic nerve transection some neurons in the L4 and L5 dorsal root ganglia begin to show immunoreactivity for nestin, a filament protein expressed by neuronal precursors and immature neurons, which is stimulated by neurotrophin-3 (NT-3) administration. The aim of this study was to examine whether NT-3 administration could be compensating for decreased production of neurotrophins or their receptors after axotomy, and to determine the effect on nestin synthesis. The levels of mRNA in the ipsilateral and contralateral L4 and L5 dorsal root ganglia were analyzed using real-time polymerase chain reaction, 1 day, 1, 2 and 4 weeks after unilateral sciatic nerve transection and NT-3 or vehicle administration via s.c. micro-osmotic pumps. In situ hybridization was used to identify which cells and neurons expressed mRNAs of interest, and the expression of full-length trkC and p75NTR protein was investigated using immunohistochemistry. Systemic NT-3 treatment increased the expression of brain-derived neurotrophic factor, nestin, trkA, trkB and trkC mRNA in ipsilateral ganglia compared with vehicle-treated animals. Some satellite cells surrounding neurons expressed trkA and trkC mRNA and trkC immunoreactivity. NT-3 administration did not affect neurotrophin mRNA levels in the contralateral ganglia, but decreased the expression of trkA mRNA and increased the expression of trkB mRNA and p75NTR mRNA and protein. These data suggest that systemically administered NT-3 may counteract the decrease, or even increase, neurotrophin responsiveness in both ipsi- and contralateral ganglia after nerve injury.

A novel role for extracellular signal-regulated kinase 5 and myocyte enhancer factor 2 in medulloblastoma cell death.

Expression of the neurotrophin-3 receptor, tyrosine kinase C (TrkC), is associated with favorable prognosis in medulloblastoma patients. This may be due to increased tumor apoptosis induced by TrkC activation. Neurotrophin-3/TrkC-induced apoptosis is inhibited by the mitogen-activated protein (MAP) kinase (MAPK) pharmacologic antagonists SB203580 and PD98059. In addition to extracellular signal-regulated kinase (ERK)-1/2, PD98059 also inhibits the more recently identified neurotrophin-responsive MAPK, ERK5 (big MAPK 1). In the present study, we investigate the contribution of ERK5 and its target myocyte enhancer factor 2 (MEF2) to neurotrophin-3/TrkC-induced medulloblastoma cell death. Neurotrophin-3 not only enhanced ERK5 phosphorylation but also significantly enhanced the transcriptional activity of MEF2, a specific target of ERK5. Overexpression of both ERK5 and MEF2 induced a statistically significant increase in cell death of neurotrophin-3-responsive and nonresponsive medulloblastoma cell lines (Daoy-trkC and Daoy) and primary cultures of patched heterozygous mouse medulloblastomas. Only those cells expressing MAP/ERK kinase 5 (MEK5) plus ERK5 or MEF2 constructs underwent apoptosis, indicating that overexpression of either is sufficient to induce medulloblastoma cell death. Expression of a dominant-negative MEF2 or small interfering RNA for the ERK5 activator, MEK5, significantly inhibited neurotrophin-3-induced cell death. The dominant-negative MEF2 construct also blocked MEK5/ERK5-induced cell death, supporting a role for MEF2 downstream of ERK5. Co-immunoprecipitation studies revealed direct interaction of phosphorylated ERK5 with MEF2 in response to neurotrophin-3. Our investigation of the mechanism of neurotrophin-3/TrkC-induced apoptosis has identified a novel role for both MEK5/ERK5 and MEF2 in cell death, suggesting that these molecules can be exploited to induce apoptosis in both TrkC-expressing and non-expressing medulloblastoma cells.

BDNF: An Oncogene or Tumor Suppressor?

Neurotrophins are a family of growth factors that are vital to the proper development of the central nervous system. Their effects on cells are governed by the expression and activation of the tyrosine kinase receptors TrkA, TrkB and TrkC. TrkB has been immensely implicated in mediating neuronal migration, development and differentiation. It has also been shown to protect several neuronal cell types from an array of cytotoxic stressors after activation by its conjugate ligand brain-derived neurotrophic factor (BDNF). Over the past two decades, it has been shown that TrkB and BDNF are up-regulated in many types of cancers, conferring aggressive phenotypes underpinned by their resistance to several standard chemotherapeutic agents. This resistance to chemotherapy is modulated by the downstream targets of the TrkB receptor which include the well-characterized PI3K /Akt growth pathway, a hallmark of uncontrolled cancer cell growth and proliferation. Pre-clinical efforts to develop inhibitors of this receptor are promising, and such inhibitors also seem to sensitize cancer cells to standard chemotherapies. However, new evidence suggests that BDNF overexpression in the hypothalamus has immunoaugmenting properties, eliciting an increased anti-tumor immune response and reducing the activity of several proteins that would normally confer resistance to chemotherapeutic agents. In the current work, we provide a global analysis of the physiological consequences of TrkB receptor activation in vitro and discuss the dynamic consequences of TrkB activation in vivo. Finally, we propose a clinically-feasible option for increasing BDNF expression in the hypothalamus to more readily utilize the oncolytic effects of BDNF.

Interferon-ß Inhibits Neurotrophin 3 Signalling and Pro-Survival Activity by Upregulating the Expression of Truncated TrkC-T1 Receptor.

Although clinically useful for the treatment of various diseases, type I interferons (IFNs) have been implicated as causative factors of a number of neuroinflammatory disorders characterized by neuronal damage and altered CNS functions. As neurotrophin 3 (NT3) plays a critical role in neuroprotection, we examined the effects of IFN-ß on the signalling and functional activity of the NT3/TrkC system. We found that prolonged exposure of differentiated human SH-SY5Y neuroblastoma cells to IFN-ß impaired the ability of NT3 to induce transphosphorylation of the full-length TrkC receptor (TrkC-FL) and the phosphorylation of downstream signalling molecules, including PLCγ1, Akt, GSK-3ß and ERK1/2. NT3 was effective in protecting the cells against apoptosis triggered by serum withdrawal or thapsigargin but not IFN-ß. Prolonged exposure to the cytokine had little effects on TrkC-FL levels but markedly enhanced the messenger RNA (mRNA) and protein levels of the truncated isoform TrkC-T1, a dominant-negative receptor that inhibits TrkC-FL activity. Cell depletion of TrkC-T1 by small interfering RNA (siRNA) treatment enhanced NT3 signalling through TrkC-FL and allowed the neurotrophin to counteract IFN-ß-induced apoptosis. Furthermore, the upregulation of TrkC-T1 by IFN-ß was associated with the inhibition of NT3-induced recruitment of the scaffold protein tamalin to TrkC-T1 and tamalin tyrosine phosphorylation. These data indicate that IFN-ß exerts a negative control on NT3 pro-survival signalling through a novel mechanism involving the upregulation of TrkC-T1.

Haploinsufficiency for trkB and trkC receptors induces cell loss and accumulation of alpha-synuclein in the substantia nigra.

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to promote survival and differentiation of midbrain dopaminergic (DAergic) neurons in vitro and in vivo. This is consistent with their expression and that of their cognate receptors, trkB and trkC, in the nigrostriatal system. Degeneration of DAergic neurons of the substantia nigra and alpha-synuclein-positive aggregates in the remaining substantia nigra (SN) neurons are hallmarks of Parkinson's disease (PD). Reduced expression of BDNF has been reported in the SN from PD patients. Moreover, mutations in the BDNF gene have been found to play a role in the development of familial PD. We show now that haploinsufficiencies of the neurotrophin receptors trkB and/or trkC cause a reduction in numbers of SN neurons in aged (21-23 month old) mice, which is accompanied by a reduced density in striatal tyrosine hydroxylase immunoreactive (TH-ir) fibers. These aged mutant mice, in contrast to wild-type littermates, display an accumulation of alpha-synuclein in the remaining TH-positive neurons of the SN. We conclude that impairment in trkB and/or trkC signaling induces a phenotype in the aged SN, which includes two hallmarks of PD, losses of TH positive neurons and axons along with massive neuronal deposits of alpha-synuclein.

The chimeric protein tyrosine kinase ETV6-NTRK3 requires both Ras-Erk1/2 and PI3-kinase-Akt signaling for fibroblast transformation.

There is increasing interest in the potential role of the NTRK family of neurotrophin receptors in human neoplasia. These receptor protein tyrosine kinases (PTKs) are well-known mediators of neuronal cell survival and differentiation, but altered NTRK signaling has also been implicated in mesenchymal, hematopoietic, and epithelial malignancies. We recently identified a novel gene fusion involving one of the neurotrophin receptor genes, NTRK3, in the pediatric solid tumor, congenital fibrosarcoma. In these tumors (and subsequently demonstrated in several other human malignancies), a t(12;15)(p13;q25) rearrangement fuses the 3' portion of the ETV6 gene with exons encoding the PTK domain of NTRK3. The resulting ETV6-NTRK3 fusion protein functions as a chimeric PTK with potent transforming activity. However, previous studies failed to detect interactions between ETV6-NTRK3 and molecules known to link wild-type NTRK3 to its two major effector pathways, namely the Ras-Raf1-Mek1-Erk1/2 mitogenic pathway or the phosphatidylinositol 3'-kinase pathway leading to activation of the AKT survival factor. Therefore, it remains unknown whether ETV6-NTRK3 transformation involves altered NTRK3 signaling. We now report that ETV6-NTRK3 expression in NIH3T3 cells leads to constitutive activation of Mek1 and Akt, as well as to constitutively high expression of cyclin D1. ETV6-NTRK3-induced soft agar colony formation was almost completely abolished by inhibition of either the Ras-Raf1-Mek1-Erk1/2 or the phosphatidylinositol 3'-kinase-Akt pathway. Moreover, this inhibition dramatically reduced expression of cyclin D1. Our results indicate that ETV6-NTRK3 transformation involves a link between known NTRK3 signaling pathways and aberrant cell cycle progression and that Mek1 and Akt activation act synergistically to mediate these effects.

Runx3-regulated expression of two Ntrk3 transcript variants in dorsal root ganglion neurons.

Somatosensation is divided into proprioception and cutaneous sensation. Dorsal root ganglion (DRG) neurons project their fibers toward peripheral targets including muscles and skin, and centrally to the spinal cord. Proprioceptive DRG neurons transmit information from muscle spindles and Golgi tendon organs to the spinal cord. We previously showed that Runt-related transcription factor 3 (Runx3) is expressed in these neurons and their projections to the ventral spinal cord and muscle spindles are lost in Runx3-deficient (Runx3(-/-) ) mouse embryos. Although Runx3 is likely to contribute to the fate decision and projection of proprioceptive DRG neurons, the precise roles for Runx3 in these phenomena are unknown. To identify genes regulated by Runx3 in embryonic DRGs, we performed microarray analyses using cDNAs isolated from wild-type and Runx3(-/-) DRGs of embryonic day (E) 12.5 and selected two transcript variants of the tyrosine kinase receptor C (TrkC) gene. These variants, Ntrk3 variant 1 (Ntrk3-v1) and variant 2 (Ntrk3-v2), encode full-length and truncated receptors of neurotrophin-3, respectively. Using double in situ hybridization, we found that most of Ntrk3-v1 mRNA expression in E14.5 DRGs depended on Runx3 but that more than half of Ntrk3-v2 mRNA one were expressed in a Runx3-independent manner. Furthermore, our data revealed that the rate of Ntrk3-v1 and Ntrk3-v2 colocalization in DRGs changed from E14.5 to E18.5. Together, our data suggest that Runx3 may play a crucial role in the development of DRGs by regulating the expression of Ntrk3 variants and that DRG neurons expressing Ntrk3-v1 but not Ntrk3-v2 may differentiate into proprioceptive ones.