TrkC Research Articles

Activity-Dependent Inhibitory Synaptogenesis in Cerebellar Cultures.

Inhibitory synapses on Purkinje cell somata in organotypic cerebellar cultures derived from newborn mice were increased after chronic exposure post explantation to agents that enhance neuronal activity. Inhibitory synaptogenesis was reduced in similar cultures after continuous blockade of spontaneous neuronal discharges. By contrast, excitatory synapses developed fully in the absence of neuronal activity. The reduction of inhibitory synaptogenesis was prevented by the simultaneous application of activity blocking agents and neurotrophins BDNF or NT-4, which are TrkB receptor ligands, but not with NT-3, a TrkC receptor ligand. The effect of endogenous neurotrophins was evaluated by continuously exposing cerebellar cultures to antibodies to BDNF and NT-4, which caused a significant reduction in the development of inhibitory Purkinje cell axosomatic synapses. These combined results indicated a role for TrkB receptors in activity-dependent inhibitory synaptogenesis. This concept was supported by the promotion of inhibitory synaptogenesis by specific antibody activation of TrkB receptors.

NTRK gene fusions as novel targets of cancer therapy across multiple tumour types

The tropomyosin receptor kinase (Trk) receptor family comprises 3 transmembrane proteins referred to as Trk A, B and C (TrkA, TrkB and TrkC) receptors that are encoded by the NTRK1, NTRK2 and NTRK3 genes, respectively. These receptor tyrosine kinases are expressed in human neuronal tissue and play an essential role in the physiology of development and function of the nervous system through activation by neurotrophins. Gene fusions involving NTRK genes lead to transcription of chimeric Trk proteins with constitutively activated or overexpressed kinase function conferring oncogenic potential. These genetic abnormalities have recently emerged as targets for cancer therapy, because novel compounds have been developed that are selective inhibitors of the constitutively active rearranged proteins. Developments in this field are being aided by next generation sequencing methods as tools for unbiased gene fusions discovery. In this article, we review the role of NTRK gene fusions across several tumour histologies, and the promises and challenges of targeting such genetic alterations for cancer therapy.

Interferon-β 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-β 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-β 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-3β and ERK1/2. NT3 was effective in protecting the cells against apoptosis triggered by serum withdrawal or thapsigargin but not IFN-β. 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-β-induced apoptosis. Furthermore, the upregulation of TrkC-T1 by IFN-β 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-β exerts a negative control on NT3 pro-survival signalling through a novel mechanism involving the upregulation of TrkC-T1.

Neurotrophin-3 Induces BMP-2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats.

Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill-hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT-3, and NT-4 and their Trk receptors. NT-3 and its receptor TrkC showed the highest induction. NT-3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT-3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT-3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP-2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT-3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP-2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT-3 osteogenic effect in vitro because it can be almost completely abrogated by co-addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT-3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co-treatment with anti-VEGF. This study suggests that NT-3 may be an osteogenic and angiogenic factor upstream of BMP-2 and VEGF in bony repair, and further studies are required to investigate whether NT-3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016 American Society for Bone and Mineral Research.

Neurotrophic factors: promising candidates in tissue regeneration.

Neurotrophic factors: promising candidates in tissue regeneration.

Abstract C187: Cross-inhibition of Trk receptors by TAS-119, a novel Aurora A selective inhibitor, exhibits therapeutic potential in a Trk-driven cancer model

Abstract Background; Comprehensive analysis by next-generation sequencing has identified oncogenic gene rearrangement of Trk neurotrophin receptors TrkA, TrkB and TrkC (encoded by NTRK1, NTRK2, and NTRK3 gene, respectively) in multiple tumor types including lung adenocarcinoma, colorectal cancer, glioblastoma and acute myeloid leukemia. The creation of gene fusion through genomic rearrangement results in constitutive activation of the kinase domain of Trk. These recent findings encourage study of the therapeutic potential of Trk kinases as drug targets for the specific treatment of patients with oncogenic NTRK gene abnormalities. TAS-119 is a novel small molecular weight inhibitor of Aurora A kinase and currently investigated in phase 1 clinical trial as both an enhancer of microtubule stabilizing drugs and monotherapy (1-3). In addition, TAS-119 shows pan-Trk inhibition with potency similar to Aurora A inhibition. Here, we describe the in vitro and in vivo pharmacologic profile of TAS-119 as a pan-Trk inhibitor. Methods; Kinase assays were performed with purified recombinant enzymes under optimized conditions using ATP concentration at or just below the respective Km. In cell proliferation studies, cell viability was determined at 72 hour by detecting cellular ATP concentration. Analysis of protein expression including phosphorylated proteins was conducted by Western blotting. In tumor xenograft models, cells were implanted subcutaneously in nude mice. TAS-119 was administered orally with twice daily or intermittent dosing (4 days on/ 3 days off schedule) for 14 days. Results; TAS-119 inhibited TrkA activity with an IC50 of 1 nM in cell-free system, and inhibited TrkA autophosphorylation and cell proliferation of KM12C colorectal cancer cell line harbouring TPM3-TrkA fusions at sub-nM range. In the KM12C xenograft model, both continuous and intermittent treatment with TAS-119 at 60 mg/kg or 100 mg/kg b.i.d. for 14 days resulted in dose-dependent inhibition of tumor growth with good tolerability. Delays of tumor growth in xenograft model were well correlated with reduction of TrkA autophosphorylation and inhibition of downstream signal transduction like pPLCγ, pERK and pAKT within tumors. Conclusions; TAS-119 is an orally bioavailable dual-specificity kinase inhibitor developed specifically for both Trk and Aurora A inhibition with favorable profiles in animals. These preclinical data suggest that TAS-119 has therapeutic potential for cancers with Trk rearrangements. Currently, phase 1 dose-escalation clinical trial is ongoing. Although TAS-119 is primarily being developed as an inhibitor for Aurora A kinase, it should also be tested clinically in patients with tumors showing Trk gene rearrangement. 1) 24th EORTC-NCI-AACR Symposium (2012), abstract #251 and #252 2) 25th EORTC-NCI-AACR Symposium (2013), abstract #A268 and #A269 3) 26th EORTC-NCI-AACR Symposium (2014), abstract #P005 and #P178 Citation Format: Hiroshi Sootome, Akihiro Miura, Kimihiro Ito, Takamasa Suzuki, Hiroshi Hirai, Teruhiro Utsugi, Kazuhiko Yonekura. Cross-inhibition of Trk receptors by TAS-119, a novel Aurora A selective inhibitor, exhibits therapeutic potential in a Trk-driven cancer model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C187.

Is there a role for neurotrophic factors and their receptors in augmenting the neuroprotective effect of (−)-epigallocatechin-3-gallate treatment of sciatic nerve crush injury?

This study analyzed and compared the effects of EGCG treatment on the expression of NTFs and NTF receptors expression in the sciatic nerve and the L3–L6 spinal cord segments at the early phase of regeneration following sciatic nerve crush injury. Analysis of BDNF, GDNF and NT3 neurotropic factors and Trk-B, Trk-C and NGFR-p75 receptors in neurons in the spinal cord of CRUSH and CRUSH + EGGC rats showed significant (p < 0.0001) decrease compared to NAÏVE and SHAM at day 1, 3, 7 and 14 after nerve injury. EGCG treatment significantly (p < 0.0001) increased the BDNF, GDN, NT3, Trk-B, Trk-C and NGFR-p75 immunostaining in the L3–L6 spinal cord compared to CRUSH animals. Also, EGCG treatment significantly increased the Trk-B protein concentration and Trk-B, NT3 and Trk-C gene expression in the spinal cords compared to CRUSH group. However, at day 1 and 3 post nerve injury, EGCG treatment significantly decreased the NGFR-p75 expression compared to CRUSH rats. In the sciatic nerve, EGCG treatment significantly (p < 0.01) increased the Trk-B and NGFR-p75 protein concentration in the controls. EGCG treatment significantly (p < 0.0001) increased the Trk-B, Trk-C and NGFR-p75 mRNA gene expressions in the sciatic nerves compared to CRUSH group. Only at day 1, CRUSH + EGCG animals displayed significant rise in the sciatic nerves NT3 gene expression compared to CRUSH group. Our data suggest that the EGCG neuroprotective effect on the spinal cord neurons may be mediated through the modulation of NTFs and NTF receptors following nerve crush injury in a rat model.

Neurotrophin-3 Enhances the Synaptic Organizing Function of TrkC-Protein Tyrosine Phosphatase σ in Rat Hippocampal Neurons.

Neurotrophin-3 (NT-3) and its high-affinity receptor TrkC play crucial trophic roles in neuronal differentiation, axon outgrowth, and synapse development and plasticity in the nervous system. We demonstrated previously that postsynaptic TrkC functions as a glutamatergic synapse-inducing (synaptogenic) cell adhesion molecule trans-interacting with presynaptic protein tyrosine phosphatase σ (PTPσ). Given that NT-3 and PTPσ bind distinct domains of the TrkC extracellular region, here we tested the hypothesis that NT-3 modulates TrkC/PTPσ binding and synaptogenic activity. NT-3 enhanced PTPσ binding to cell surface-expressed TrkC and facilitated the presynapse-inducing activity of TrkC in rat hippocampal neurons. Imaging of recycling presynaptic vesicles combined with TrkC knockdown and rescue approaches demonstrated that NT-3 rapidly potentiates presynaptic function via binding endogenous postsynaptic TrkC in a tyrosine kinase-independent manner. Thus, NT-3 positively modulates the TrkC-PTPσ complex for glutamatergic presynaptic assembly and function independently from TrkC kinase activation. Our findings provide new insight into synaptic roles of neurotrophin signaling and mechanisms controlling synaptic organizing complexes. Significance statement: Although many synaptogenic adhesion complexes have been identified in recent years, little is known about modulatory mechanisms. Here, we demonstrate a novel role of neurotrophin-3 in synaptic assembly and function as a positive modulator of the TrkC-protein tyrosine phosphatase σ complex. This study provides new insight into the involvement of neurotrophin signaling in synapse development and plasticity, presenting a molecular mechanism that may underlie previous observations of short- and long-term enhancement of presynaptic function by neurotrophin. Given the links of synaptogenic adhesion molecules to autism and schizophrenia, this study might also contribute to a better understanding of the pathogenesis of these disorders and provide a new direction for ameliorating imbalances in synaptic signaling networks.

Current outcome predictors in childhood primitive neuroectodermal tumors

Central nervous system primitive neuroectodermal tumors (PNET) including cerebellar PNET (medulloblastoma, PNET/MB) are the most common malignant brain tumors in childhood. Because of the high risk of leptomeningeal dissemination, standard postoperative treatment for PNET includes craniospinal radiotherapy and chemotherapy. Such treatment causes long-term morbidity including endocrine and growth disturbances, as well as neurocognitive dysfunction, which is particularly severe in young children. Over the last years, neurotrophin receptor TrkC, neuregulin receptor ErbB2 and oncogene MYC have been identified as biological prognostic factors in PNET/MB. High TrkC mRNA expression, low ErbB2 protein expression and low MYC mRNA expression are powerful independent predictors of favorable clinical outcome in PNET/MB. Upon validation in ongoing prospective biological studies, these biological factors may define risk groups and help direct therapy decisions for children with PNET/MB. In PNET with favorable biological factors and no evidence of leptomeningeal tumor dissemination, therapy with reduced craniospinal radiation might retain the efficacy but reduce the toxicity and therefore improve the quality of life for the survivors. (J Pediatr Neurol 2003; 1(2): 75–82).

Deletion of a kinesin I motor unmasks a mechanism of homeostatic branching control by neurotrophin-3.

Development and function of highly polarized cells such as neurons depend on microtubule-associated intracellular transport, but little is known about contributions of specific molecular motors to the establishment of synaptic connections. In this study, we investigated the function of the Kinesin I heavy chain Kif5aa during retinotectal circuit formation in zebrafish. Targeted disruption of Kif5aa does not affect retinal ganglion cell differentiation, and retinal axons reach their topographically correct targets in the tectum, albeit with a delay. In vivo dynamic imaging showed that anterograde transport of mitochondria is impaired, as is synaptic transmission. Strikingly, disruption of presynaptic activity elicits upregulation of Neurotrophin-3 (Ntf3) in postsynaptic tectal cells. This in turn promotes exuberant branching of retinal axons by signaling through the TrkC receptor (Ntrk3). Thus, our study has uncovered an activity-dependent, retrograde signaling pathway that homeostatically controls axonal branching.

Neurotrophin Signaling via TrkB and TrkC Receptors Promotes the Growth of Brain Tumor-initiating Cells

Neurotrophins and their receptors are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However, the role of Trk receptors has not been examined. In this study, we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here, we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC, not TrkA, and they also express neurotrophins NGF, BDNF, and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further, pharmacological inhibition of both ERK and Akt pathways blocked BDNF, and NT3 stimulated BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling, and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma.

Dehydroepiandrosterone: an ancestral ligand of neurotrophin receptors.

Dehydroepiandosterone (DHEA), the most abundant steroid in humans, affects multiple cellular functions of the endocrine, immune, and nervous systems. However, up to quite recently, no receptor has been described specifically for it, whereas most of its physiological actions have been attributed to its conversion to either androgens or estrogens. DHEA interacts and modulate a variety of membrane and intracellular neurotransmitter and steroid receptors. We have recently reported that DHEA protects neuronal cells against apoptosis, interacting with TrkA, the high-affinity prosurvival receptor of the neurotrophin, nerve growth factor. Intrigued by its pleiotropic effects in the nervous system of a variety of species, we have investigated the ability of DHEA to interact with the other two mammalian neurotrophin receptors, ie, the TrkB and TrkC, as well as their invertebrate counterparts (orthologs) in mollusks Lymnaea and Aplysia and in cephalochordate fish Amphioxus. Amazingly, DHEA binds to all Trk receptors, although with lower affinity by 2 orders of magnitude compared with that of the polypeptidic neurotrophins. DHEA effectively induced the first step of the TrkA and TrkC receptors activation (phosphorylation at tyrosine residues), including the vertebrate neurotrophin nonresponding invertebrate Lymnaea and Aplysia receptors. Based on our data, we hypothesize that early in evolution, DHEA may have acted as a nonspecific neurotrophic factor promoting neuronal survival. The interaction of DHEA with all types of neurotrophin receptors offers new insights into the largely unidentified mechanisms of its actions on multiple tissues and organs known to express neurotrophin receptors.

TRKing down an old oncogene in a new era of targeted therapy.

The use of high-throughput next-generation sequencing techniques in multiple tumor types during the last few years has identified NTRK1, 2, and 3 gene rearrangements encoding novel oncogenic fusions in 19 different tumor types to date. These recent developments have led us to revisit an old oncogene, Trk (originally identified as OncD), which encodes the TPM3-NTRK1 gene fusion and was one of the first transforming chromosomal rearrangements identified 32 years ago. However, no drug has yet been approved by the FDA for cancers harboring this oncogene. This review will discuss the biology of the TRK family of receptors, their role in human cancer, the types of oncogenic alterations, and drugs that are currently in development for this family of oncogene targets. Precision oncology approaches have accelerated recently due to advancements in our ability to detect oncogenic mutations in tumor samples. Oncogenic alterations, most commonly gene fusions, have now been detected for the genes encoding the TRKA, TRKB, and TRKC receptor tyrosine kinases across multiple tumor types. The scientific rationale for the targeting of the TRK oncogene family will be discussed here.

TAM receptors support neural stem cell survival, proliferation and neuronal differentiation.

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III+) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.

Selective conversion of fibroblasts into peripheral sensory neurons.

Humans and mice detect pain, itch, temperature, pressure, stretch and limb position via signaling from peripheral sensory neurons. These neurons are divided into three functional classes (nociceptors/pruritoceptors, mechanoreceptors and proprioceptors) that are distinguished by their selective expression of TrkA, TrkB or TrkC receptors, respectively. We found that transiently coexpressing Brn3a with either Ngn1 or Ngn2 selectively reprogrammed human and mouse fibroblasts to acquire key properties of these three classes of sensory neurons. These induced sensory neurons (iSNs) were electrically active, exhibited distinct sensory neuron morphologies and matched the characteristic gene expression patterns of endogenous sensory neurons, including selective expression of Trk receptors. In addition, we found that calcium-imaging assays could identify subsets of iSNs that selectively responded to diverse ligands known to activate itch- and pain-sensing neurons. These results offer a simple and rapid means for producing genetically diverse human sensory neurons suitable for drug screening and mechanistic studies.

Establishment of a cellular model to study TrkC-dependent neuritogenesis.

The rat PC12 cell line has become a widely used research tool for many aspects of neurobiology. Nerve growth factor (NGF)-responsive PC12 cells were engineered to drive expression of doxycycline (Dox)-induced gene of interest in the Tet-On expression system that resulted in obtaining PC12-Tet-On cells. TrkA and TrkC are neurotrophin receptors derived from the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases. TrkA receptor binds and is activated mainly by NGF, while TrkC receptor binds and is activated by neurotrophin 3 (NT3). The purpose of this research was to design and describe PC12-based neuronal cell model to study TrkC-triggered versus TrkA-triggered neurite outgrowth. The second-generation tetracycline-responsive promoter (P tight) was used in order to provide low basal expression in the absence of Dox and high-level Dox-induced expression of TrkC. The main advantage of presented model system is dependence of TrkC level on Dox concentration. It also allows to compare activation of intracellular signaling proteins and neurite outgrowth following activation of TrkA and TrkC receptors by NGF and NT3, respectively, in the context of the same quality and quantity of intracellular adaptor proteins, Ras proteins, protein kinases and phosphatases, and phospholipase Cγ1, as a difference in the activation of intracellular signaling network by these two distinct although related receptor tyrosine kinases is expected. The results of our studies suggest that despite slightly weaker activation of ERK1/2 mitogen-activated protein kinases, NT3-triggered TrkC seems to provide apparently stronger than NGF-triggered TrkA signal for neurite elongation in differentiating PC12 cells.

CS-14 * NEUROTROPHIN RECEPTORS TrkB AND TrkC ARE REQUIRED FOR SURVIVAL OF BRAIN TUMOR INITIATING CELLS

Neurotrophins (NGF, BDNF and NT3) and their receptors (TrkA, TrkB, TrkC and p75NTR) are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75NTR is required for glioma invasion and proliferation. However, the role of other Trk receptors has not been examined. In this study, we investigated the importance of neurotrophin receptors TrkB and TrkC in survival of brain tumor initiating cells (BTICs). We used human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas and examined the expression of neurotrophin receptors and studied their role in survival/growth these cells in vitro, we have also examined the mechanism underlie. Here, we show that human malignant glioma tissues and tumor-initiating cells express the neurotrophin receptors TrkB and TrkC, not TrkA and they also express neurotrophins NGF, BDNF and NT3. Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 stimulates Trk phosphorylation, activates Erk and Akt and enhances tumor-initiating cell viability and growth. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition using inhibitors of Trk, Erk and Akt, decreases neurotrophin-dependent Trk phosphorylation, Erk and Akt activation and BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling and further neurotrophin signaling is required for maintains long-term BTIC growth in the absence of EGF and FGF, and this effect is attenuated with Trk, Erk and Akt inhibitors. In vivo experiments are under progress. Our results highlight a novel role for neurotrophin signaling in malignant glioma and suggest that Trks could be a target for combinatorial treatment.

Graft of the gelatin sponge scaffold containing genetically-modified neural stem cells promotes cell differentiation, axon regeneration, and functional recovery in rat with spinal cord transection.

Biological materials combined with genetically-modified neural stem cells (NSCs) are candidate therapy targeting spinal cord injury (SCI). Based on our previous studies, here we performed gelatin sponge (GS) scaffold seeded with neurotrophin-3 (NT-3) and its receptor TrkC gene modifying NSCs for repairing SCI. Eight weeks later, compared with other groups, neurofilament-200 and 5-hydroxytryptamine positive nerve fibers were more in the injury site of the N+T-NSCs group. Immunofluorescence staining showed the grafted NSCs could differentiate into microtubule associated protein (Map2), postsynaptic density (PSD95), and mouse oligodendrocyte special protein (MOSP) positive cells. The percentage of the Map2, PSD95, and MOSP positive cells in the N+T-NSCs group was higher than the other groups. Immuno-electron microscopy showed the grafted NSCs making contact with each other in the injury site. Behavioral analysis indicated the recovery of hindlimbs locomotion was better in the groups receiving cell transplant, the best recovery was found in the N+T-NSCs group. Electrophysiology revealed the amplitude of cortical motor evoked potentials was increased significantly in the N+T-NSCs group, but the latency remained long. These findings suggest the GS scaffold containing genetically-modified NSCs may bridge the injury site, promote axon regeneration and partial functional recovery in SCI rats.

Peripheral nerve injury modulates neurotrophin signaling in the peripheral and central nervous system.

Peripheral nerve injury disrupts the normal functions of sensory and motor neurons by damaging the integrity of axons and Schwann cells. In contrast to the central nervous system, the peripheral nervous system possesses a considerable capacity for regrowth, but regeneration is far from complete and functional recovery rarely returns to pre-injury levels. During development, the peripheral nervous system strongly depends upon trophic stimulation for neuronal differentiation, growth and maturation. The perhaps most important group of trophic substances in this context is the neurotrophins (NGF, BDNF, NT-3 and NT-4/5), which signal in a complex spatial and timely manner via the two structurally unrelated p75(NTR) and tropomyosin receptor kinase (TrkA, Trk-B and Trk-C) receptors. Damage to the adult peripheral nerves induces cellular mechanisms resembling those active during development, resulting in a rapid and robust increase in the synthesis of neurotrophins in neurons and Schwann cells, guiding and supporting regeneration. Furthermore, the injury induces neurotrophin-mediated changes in the dorsal root ganglia and in the spinal cord, which affect the modulation of afferent sensory signaling and eventually may contribute to the development of neuropathic pain. The focus of this review is on the expression patterns of neurotrophins and their receptors in neurons and glial cells of the peripheral nervous system and the spinal cord. Furthermore, injury-induced changes of expression patterns and the functional consequences in relation to axonal growth and remyelination as well as to neuropathic pain development will be reviewed.

Dual Innervation of Neonatal Merkel Cells in Mouse Touch Domes

Merkel cell-neurite complexes are specialized mechanosensory end organs that mediate discriminative touch sensation. It is well established that type I slowly adapting (SAI) mechanoreceptors, which express neural filament heavy chain (NFH), innervate Merkel cells. It was previously shown that neurotrophic factor NT3 and its receptor TrkC play crucial roles in controlling touch dome Merkel cell innervation of NFH+ fibers. In addition, nerve fibers expressing another neurotrophic tyrosine receptor kinase (NTRK), Ret, innervate touch dome Merkel cells as well. However, the relationship between afferents responsive to NT3/TrkC signaling and those expressing Ret is unclear. It is also controversial if these Ret+ fibers belong to the early or late Ret+ DRG neurons, which are defined based on the co-expression and developmental dependence of TrkA. To address these questions, we genetically traced Ret+ and TrkC+ fibers and analyzed their developmental dependence on TrkA. We found that Merkel cells in neonatal mouse touch domes receive innervation of two types of fibers: one group is Ret+, while the other subset expresses TrkC and NFH. In addition, Ret+ fibers depend on TrkA for their survival and normal innervation whereas NFH+ Merkel cell innervating fibers are almost unaltered in TrkA mutant mice, supporting that Ret+ and NFH+/TrkC+ afferents are two distinct groups. Ret signaling, on the other hand, plays a minor role for the innervation of neonatal touch domes. In contrast, Merkel cells in the glabrous skin are mainly contacted by NFH+/TrkC+ afferents. Taken together, our results suggest that neonatal Merkel cells around hair follicles receive dual innervation while Merkel cells in the glabrous skin are mainly innervated by only SAI mechanoreceptors. In addition, our results suggest that neonatal Ret+ Merkel cell innervating fibers most likely belong to the late but not early Ret+ DRG neurons.