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PubMed1996

Synchronous onset of NGF and TrkA survival dependence in developing dorsal root ganglia.

Determinations of dorsal root ganglion (DRG) neuron loss in nerve growth factor (NGF) and neurotrophin-3 (NT-3) null mutant mice have supported the concept that neurons can switch neurotrophin dependence by revealing that many neurons must require both of these factors acting either sequentially or simultaneously during development. The situation is complex, however, in that NT-3(-/-) mutant mice show far greater neuron loss than mice deficient in the NT-3 receptor TrkC, suggesting that NT-3 may support many DRG neurons via actions on the NGF receptor TrkA. To assess the possibility of ligand-receptor cross-talk as a developmental mechanism, we have compared the onset of survival dependence of lumbar DRG neurons on NT-3, TrkC, NGF, and TrkA signaling in mice deficient in these molecules as a result of gene targeting. At embryonic day 11.5 (E11.5), virtually all lumbar DRG cells express TrkC mRNA and many require NT-3 and TrkC signaling for survival. In contrast, although many lumbar DRG cells also express TrkA at E11.5, there is little survival dependence on TrkA signaling. By E13.5, most lumbar DRG cells have downregulated TrkC mRNA. The onset of survival dependence on NGF and TrkA-signaling is concurrent and of equal magnitude at E13.5, demonstrating that NT-3 alone does not support DRG neurons via TrkA, nor can NT-3 compensate for the loss of NGF. We conclude that many murine DRG cells require NT-3 activation of TrkA is unimportant to these early NT-3 survival-promoting actions. We suggest that the discrepancy in cell loss between NT-3(-/-) and trkC(-/-) mutants is attributable to the ability of NT-3 to support DRG neurons via TrkA in the artificial situation where TrkC is absent.

Animals↗
PubMed1999

Differences in signaling properties of the cytoplasmic domains of the insulin receptor and insulin-like growth factor receptor in 3T3-L1 adipocytes.

Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase.

3T3 Cells↗
PubMed2000

Role of neurotropins in rat embryonic testis morphogenesis (cord formation).

The process of seminiferous cord formation is the first morphological event that differentiates a testis from an ovary and indicates male sex determination. Cord formation occurs by embryonic Day 14 (Day 0 = plug date; E14) in the rat. A series of experiments were conducted to determine if neurotropins and their receptors are important for the process of rat embryonic cord formation. The expression of low affinity neurotropin receptor (p75/LNGFR) was determined by immunohistochemistry on sections of both testis and ovary from E13 through birth (Day 0, P0) with an antibody to p75/LNGFR. The staining for p75/LNGFR was present in the mesonephros of E13 gonads and in a sex-specific manner appeared around developing cords at E14 in the embryonic testis. At birth, staining for p75/LNGFR was localized to a single layer of cells (i.e., peritubular cells) that surrounded the seminiferous cords. The genes for both neurotropin 3 (NT3) and for corresponding high affinity neurotropin trkC receptor were found to be expressed in the E14 rat testis, as well as other neurotropins and receptors. Immunocytochemical analysis of E14 rat testis demonstrated that NT3 was localized to the Sertoli cells and trkC was present in individual cells of the interstitium at E16 and in selected preperitubular cells at E18. Previously, the peritubular cells adjacent to the cords were demonstrated to be derived from migrating mesonephros cells around the time of cord formation. To determine if neurotropins were involved in cord formation, the actions of neurotropins were inhibited. A high affinity neurotropin receptor (trk)-specific kinase inhibitor, K252a, was used to treat organ cultures of testes from E13 rats prior to cord formation. Treatment of E13 testis organ cultures with K252a completely inhibited cord formation. K252a-treated organ cultures of E14 testis that contained cords did not alter cord morphology. A second experiment to inhibit neurotropin actions utilized a specific antagonist trk-IgG chimeric fusion protein and E13 testis organ cultures. The trk-IgG molecules dimerize with endogenous trk receptors and inhibit receptor signaling and activation of ligand function. Forty percent of E13 testis organ cultures treated with trkC-IgG had significantly reduced cord formation. TrkA-IgG had no effect on initiation of cords; however, in fifty percent of the treated organs, a "swollen" appearance of the cord structures was observed. Experiments using trkB-IgG chimeric protein on E13 organ cultures had no effect on cord formation or cord morphology. The testes from trkC and NT3 knockout mice were examined to determine if there were any morphological differences in the testis. NT3 knockouts appeared to have normal cord morphology in E15 and E17 testis. TrkC knockout mice also had normal cord morphology in E14 and P0 testis. Both NT3 and trkC knockout-mice testis had less interstitial area than wild-type controls. In addition, the trkC knockout mice have an increased number of cells expressing p75LNGFR within the cords when compared to controls or NT3 knockout mice. Combined observations suggest compensation between the different neurotropin ligands, receptors, and/or possibly different growth factors for this critical biological process. In summary, results suggest a novel nonneuronal role for neurotropins in the process of cord formation during embryonic rat testis development. The hypothesis developed is that neurotropins are involved in the progression of male sex differentiation and are critical for the induction of embryonic testis cord formation.

Animals↗
PubMed1993

Differential expression of neurotrophin receptors during renal development.

Early kidney differentiation is driven by local cell-cell interactions. The metanephrogenic mesenchyme stimulates the epithelial ureter bud to grow and branch, whereas the ureter bud stimulates the mesenchyme to convert into a new epithelium. These interactions may be dependent on local growth factors and their receptors. We studied the expression of receptors for nerve growth factors during kidney development. Expression of the low- and high-affinity receptors was cell-type specific. The low-affinity NGF receptor was found in the uninduced mesenchyme at early developmental stages, but in the glomerular podocytes at later developmental stages. In contrast, the high-affinity trkB receptor was found in the cortical mesenchyme cells that will differentiate into stroma. The trkC receptor was found only weakly expressed and in a few parts of the collecting ducts. The role of these receptors and c-ros, a receptor-type kinase expressed on the tip of the ureter bud, was studied by modified antisense oligonucleotides. However, we found that both sense, antisense and nonsense phosphorothioate oligonucleotides inhibited mouse and rat embryonic kidney development in vitro. The oligonucleotides appeared to be toxic for rodent embryonic kidneys in the experimental conditions that we used. Moreover, oligonucleotides did not penetrate well into the epithelial sheets in the organ cultures. We conclude that studies with phosphorothioate antisense oligonucleotides in organ cultures of embryonic kidneys should be interpreted with caution. Our current data do not allow us to not assign a function for the low- or high-affinity NGF receptors or c-ros in kidney development.

Animals↗
PubMed2002

Classifying the medulloblastoma: insights from morphology and molecular genetics.

Significant advances in the treatment of the medulloblastoma (MB) have been made in the last 30 years, reducing mortality by 2-fold. Further improvements in the cure rate require an increased understanding of the biology of MBs, and this will translate into refinements in their classification. Scrutiny of the cytological variation found among MBs has recently led to the concept of the anaplastic MB, which overlaps the large-cell variant and appears to share its poor prognosis. In contrast, the MB with extensive nodularity, a distinctive nodular/desmoplastic variant occurring in infants, has a better outcome than most MBs in these young patients. Building on cytogenetic studies that have drawn attention to abnormalities on chromosome 17 in over a third of MBs, research shows non-random losses on chromosomes 8, 9, 10, 11 and 16, and gains on chromosomes 1, 7 and 9. Overexpression of ErbB2 receptors and losses on chromosome 17p have been proposed as independent indicators of aggressive behaviour, while high TrkC receptor expression indicates a favourable outcome. There is a strong association between anaplastic/large-cell tumours and MYC amplification, which has previously been linked with aggressive disease, but associations between abnormalities on chromosome 17 and anaplastic/large-cell MBs and between abnormalities in the shh/PTCH pathway and the desmoplastic variant are more controversial. Classification of the MB histopathologically and according to profiles of molecular abnormalities will help both to rationalize approaches to therapy, increasing the cure rate and reducing long-term side-effects, and to suggest novel treatments.

Cerebellar Neoplasms↗
PubMed1996

Change in neurotrophins and their receptor mRNAs in the rat forebrain after status epilepticus induced by pilocarpine.

We studied the effects of status epilepticus (SE) induced by lithium chloride/pilocarpine treatment on gene expression of neurotrophins of the nerve growth factor (NGF) family and of their high-affinity receptors of the tyrosine protein kinase (trk) family in the forebrain. Using in situ hybridization (ISH), we demonstrated an early (3 h after treatment) increase in brain-derived neurotrophic factor (BDNF) and trkB mRNA expression in the dentate gyrus, amygdala, and piriform cortex, as well as widespread increases in the cerebral cortex. NGF mRNA, but not the mRNA of its receptor trkA, was increased in the dentate gyrus. In contrast, 12 h after treatment, neurotrophin-3 (NT-3) decreased, and its receptor trkC mRNA increased. There was no change in NT-4 mRNA levels. All changes were blocked by pretreatment with scopolamine, a muscarinic antagonist. The noncompetitive N-methyl-D-aspartate (NMDA) antagonist ketamine blocked NGF, BDNF, and trkB mRNA increases in the hippocampus and cerebral cortex, but not in the amygdala and piriform cortex. In contrast, ketamine did not affect NT-3 and trkC changes. These results provide a complete description of changes in mRNA levels of neurotrophins and their receptors in the forebrain after SE and supply additional data supporting the view that neurotrophin gene expression is related to abnormal neuronal activity.

Animals↗
PubMed1996

Development of striatal dopaminergic function. III: Pre- and postnatal development of striatal and cortical mRNAs for the neurotrophin receptors trkBTK+ and trkC and their regulation by synaptic dopamine.

Known neurotrophins in the nigrostriatal system include brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) which exert biological effects after high affinity binding to their respective tyrosine kinase receptors, trkB and trkC. We measured striatal trkBTK+ and trkC mRNAs in rat brain sections with in situ hybridization histochemistry. Both trkBTK+ and trkC transcripts were present in the striatal anlage at embryonic day (E) 14 or 16. Striatal trkBTK+ mRNA levels increased to a peak in the late prenatal period and gradually declined in the postnatal period. In contrast, striatal trkC mRNA levels peaked on E16, then declined to fairly constant levels. Striatal trkBTK+ gene expression increased from the medial to lateral quadrants throughout development, whereas trkC mRNA increased from the lateral to medial quadrants prenatally but increased from the dorsal to ventral quadrants on postnatal days (P) 1 and 3. The distinct spatiotemporal developmental profiles of trkBTK+ and trkC mRNA suggest that their respective ligands BDNF and NT-4/5, and NT-3, may have specialized functions in striatal neuronal development. Because neurotransmitters may regulate neurotrophin function in developing neural systems, we treated rats of various ages with the indirect dopamine agonist cocaine and measured the effects of cocaine treatment on transcription of the trk genes. Acute 1 h cocaine treatment increased trkBTK+ and trkC mRNA levels in the P5 striatum but not in the E15, E20, or adult striatum. The trkBTK+ effect was blocked by pretreatment with the D1 receptor antagonist, SCH23390, and was not affected by pretreatment with the D2 receptor antagonist, eticlopride. In contrast, trkC regulation may be mediated by independent stimulation of D1 and D2 receptors. We hypothesize that the endogenous nigrostriatal neurotransmitter dopamine can modulate striatal neurotrophin responsiveness and thereby influence striatal neuronal development during a defined developmental period.

Animals↗
PubMed1994

Role of variable beta-hairpin loop in determining biological specificities in neurotrophin family.

The neurotrophins are members of a family of structurally and functionally related neurotrophic factors that control the development and maintenance of vertebrate neurons. The crystal structure of nerve growth factor (NGF), the prototypic member of this family, contains three pairs of anti-parallel beta-strands connected by beta-hairpin loops, which contain most of the variable residues among the four neurotrophin proteins. Recently, amino acid residues in these variable loop regions have been implicated in the interaction between NGF and its signal-transducing receptor TrkA. In NGF, residues 40-49 (variable region II) span a very flexible and solvent-accessible beta-hairpin loop that is highly variable between different neurotrophins. To investigate the role of this domain in determining biological specificities in the neurotrophin family, we constructed a series of chimeric molecules by exchanging this variable region among three neurotrophins (i.e. NGF, neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4)) that bind and activate three different Trk receptors (i.e. TrkA, -C, and -B, respectively). The chimeric molecules were analyzed for their ability to activate different Trk receptor subtypes and to promote the survival of subpopulations of peripheral neurons expressing specific types of Trk mRNAs. Exchange of the 40-49 variable loop region between NGF and NT-3 resulted in molecules capable of activating both TrkA and TrkC receptors and of rescuing neurons containing TrkA and TrkC mRNAs, indicating that this loop plays an important role in determining biological specificities in these two neurotrophins. Furthermore, variable region II from NT-4 conferred the ability to differentiate nnr5 PC12-TrkB cells to a chimeric NT-3 molecule that was originally incapable of eliciting a response in these cells. In contrast, exchanges between NGF and NT-4 did not suffice to generate molecules with broader biological specificities, suggesting that other regions in these molecules are also required. Our results support the evolutionary relationships between the three polypeptides deduced from structural comparisons.

3T3 Cells↗
PubMed1997

Analysis of neurotrophin/receptor interactions with a gD-flag-modified quantitative kinase receptor activation (gD.KIRA) enzyme-linked immunosorbent assay.

A rapid, sensitive, and high-capacity assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a "kinase receptor activation" or KIRA-ELISA, utilizes two separate microtiter plates, one for cell culture and ligand stimulation, and the other for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of neurotrophin-induced trkA, trkB, or trkC activation. It utilizes a trkA, trkB, or trkC receptor fused with a 26-amino-acid polypeptide flag derived from HSV glycoprotein D (gD.trkA, B, or C, respectively) on the amino-terminus, stably transfected into CHO cells. Stimulated receptors were solubilized with Triton X-100 buffer and then captured in ELISA wells coated with gD-specific mAb. The degree of receptor autophosphorylation was quantified by anti-phosphotyrosine ELISA. Reproducible standard curves were generated with an EC50 of approximately 16 ng/ml NGF for gD.trkA KIRA, 11 ng/ml for NT4/5 and 20 ng/ml for BDNF in gD.trkB KIRA, and 9.4 ng/ml for NT3 in gD.trkC KIRA. When the gD.trkA KIRA assay was used to quantify serum NGF or NT3 following administration to rats, the assay agreed well with currently existing ELISA assays. When the gD.trkA KIRA assay was used to test several NGF variants, as well as NGF stability samples, the capacity of the assay to quantify ligand bioactivity compared well with the more widely used radioreceptor binding and PC 12 cell survival assays. The gD.trk KIRA assays show great potential as rapid bioassays, capable of quantitative, consistent, and stability indicating analyses.

Animals↗
PubMed2005

Ras activation of a Rac1 exchange factor, Tiam1, mediates neurotrophin-3-induced Schwann cell migration.

Endogenous neurotrophins positively and negatively regulate migration of premyelinating Schwann cells before the initiation of myelination. Neurotrophin-3 (NT3) acting through the TrkC receptor tyrosine kinase stimulates Schwann cell migration via the Rho GTPases Rac1 and Cdc42. We previously demonstrated that TrkC directly phosphorylates and activates Dbs, the guanine-nucleotide exchange factor (GEF) for Cdc42, to partially mediate Schwann cell migration. Here, we identify T lymphoma invasion and metastasis (Tiam) 1 as the Rac1-specific guanine-nucleotide exchange factor involved in NT3-induced Schwann cell migration. Furthermore, the interaction between the small GTPase Ras and Tiam1 plays an essential role in the activation of Rac1. Taken together, these results suggest that NT3 activation of TrkC stimulates Schwann cell migration through two parallel signaling units, Ras/Tiam1/Rac1 and Dbs/Cdc42, and that Schwann cell migration is uniquely regulated in the case of Ras and Rac1, by two different types of small GTPases.

Analysis of Variance↗
PubMed1997

The role of neurotrophic factors in regulating the development of inner ear innervation.

Several neurotrophins and their receptors regulate the survival of vestibular and cochlear neurons and probably also the efferent and autonomic neurons that innervate the inner ear. Mice lacking either brain-derived neurotrophic factor (BDNF) or its associated receptor, TrkB, lose all innervation to the semicircular canals and have reduced innervation of the outer hair cells in the apical and middle turns of the cochlea. Mice lacking neurotrophin-3 (NT-3) or its receptor, TrkC, lose many spiral ganglion cells predominantly in the basal turn of the cochlea. Nerve fibers from spiral ganglion cells in the middle turn extended to inner hair cells of the base. In mice lacking both BDNF and NT-3, or both TrkB and TrkC, there is a complete loss of innervation to the inner ear. Thus, these two neurotrophins and their associated receptors have been shown to be absolutely necessary for the normal development of afferent innervation of the inner ear. Current research efforts are testing the therapeutic potential for neurotrophins to treat hearing loss.

Animals↗
PubMed2003

TrkB receptor signaling and activity-dependent inhibitory synaptogenesis.

When mouse organotypic cerebellar cultures were exposed to anti-GABA agents that increased neuronal activity early in development, there was a doubling of the ratio of inhibitory axosomatic synapse profiles to Purkinje cell somatic profiles after two weeks in vitro, which correlated with a decrease in spontaneous cortical discharges. When similar cultures were maintained in medium with activity blocking agents, Purkinje cell axosomatic synapses were reduced to approximately half of control values and, after recovery from activity blockade, the cultures discharged hyperactively. By contrast, the full complement of excitatory cortical synapses developed in the absence of neuronal activity. These results support the concept that neuronal activity is necessary for the complete development of inhibitory circuitry. When cerebellar cultures were simultaneously exposed to activity blocking agents and to neurotrophins BDNF or NT-4, both of which bound to the TrkB receptor, the numbers of inhibitory Purkinje cell axosomatic synapses were similar to those of untreated control cultures, and control rates of spontaneous cortical discharges were recorded. The TrkC receptor ligand, NT-3, did not promote inhibitory synapse development in the absence of neuronal activity, and such cultures exhibited hyperactive cortical discharges. These results are consistent with a role for TrkB receptor ligands in activity-dependent inhibitory synaptogenesis. Subsequent exposure of cerebellar cultures to antibody to the extracellular domain of TrkB induced an increased development of Purkinje cell axosomatic synapses, while similar antibody activation of TrkC had no effect on inhibitory synaptogenesis. The promotion of inhibitory synapse development by specific antibody activation of TrkB supports the concept that signaling for activity-dependent inhibitory synaptogenesis is via the TrkB receptor.

Animals↗
PubMed1998

The ras-related GTPase rac1 regulates a proliferative pathway selectively utilized by G-protein coupled receptors.

Ras and rac are each members of the superfamily of monomeric GTPases and both function as molecular switches to link cell-surface signals to intracellular responses. Using a novel assay of cellular proliferation called R-SAT (Receptor Selection and Amplification Technology), we examined the roles of ras and rac in mediating the proliferative responses to a variety of cell-surface receptors. Activated, wild-type and dominant-negative mutants of rac and ras were tested for their effects on cellular proliferation either alone or in combination with receptors. Activated rac (rac Q61L, henceforth rac*) and ras (ras G12V, henceforth ras*) each induced strong proliferative responses. Dominant-negative rac (rac T17N, henceforth rac(-)) dramatically suppressed proliferative responses to G-protein coupled receptors (GPCR's) including the m5 muscarinic receptor and the alpha1B adrenergic receptor. In contrast, rac(-) had little or no effect upon responses to the tyrosine kinase receptor TrkC, and only partially suppressed responses to the Janus kinase (JAK/STAT) linked granulocyte macrophage colony stimulating factor (GM-CSF) receptor. Dominant-negative ras (ras T17N, henceforth ras(-)) blocked the proliferative responses to all of the tested receptors. Compared to rac(-) and ras(-), wild-type rac and ras had only modest effects on the tested receptors. Overall these results demonstrate that rac mediates the proliferative effects of G-protein coupled receptors through a pathway that is distinct from the proliferative signaling pathway utilized by tyrosine kinase linked and JAK-linked receptors.

3T3 Cells↗
PubMed1997

K252a inhibits proliferation of glioma cells by blocking platelet-derived growth factor signal transduction.

Growth factors are known to regulate glioma proliferation. The glioma cell lines U87 and T98G were examined for evidence of an autocrine stimulatory loop involving the neurotrophin family of growth factors. Although neurotrophin-3 and TrkC RNA were detected by reverse transcription-PCR, there was no evidence of significant interaction between neurotrophin-3 and its cognate receptor TrkC. The microbial alkaloid K252a has been described to inhibit both Trk tyrosine kinase activity and neuroblastoma cell proliferation. K252a inhibited proliferation in U87 (IC50 = 1170 nM) and T98G (IC50 = 529 nM) but induced apoptosis in U87 cells only. At concentrations of 500 nM to 1 microM, K252a blocked only platelet-derived growth factor (PDGF)-mediated receptor autophosphorylation. These results suggest that an autocrine loop involving PDGF is functional and important for maintaining tumor growth. There is no evidence to support the existence of a neurotrophin-mediated autocrine loop. K252a, through inhibition of PDGF signal transduction, may be a novel therapeutic agent in the treatment of human gliomas.

Apoptosis↗
PubMed2005

SLAM-associated protein as a potential negative regulator in Trk signaling.

Neurotrophin signaling plays important roles in regulating the survival, differentiation, and maintenance of neurons in the nervous system. Binding of neurotrophins to their cognate receptors Trks induces transactivation and phosphorylation of the receptor at several tyrosine residues. These phosphorylated tyrosine residues then serve as crucial docking sites for adaptor proteins containing a Src homology 2 or phosphotyrosine binding domain, which upon association with the receptor initiates multiple signaling events to mediate the action of neurotrophins. Here we report the identification of a Src homology 2 domain-containing molecule, SLAM-associated protein (SAP), as an interacting protein of TrkB in a yeast two-hybrid screen. SAP was initially identified as an adaptor molecule in SLAM family receptor signaling for regulating interferon-gamma secretion. In the current study, we found that SAP interacted with TrkA, TrkB, and TrkC receptors in vitro and in vivo. Binding of SAP required Trk receptor activation and phosphorylation at the tyrosine 674 residue, which is located in the activation loop of the kinase domain. Overexpression of SAP with Trk attenuated tyrosine phosphorylation of the receptors and reduced the binding of SH2B and Shc to TrkB. Moreover, overexpression of SAP in PC12 cells suppressed the nerve growth factor-dependent activation of extracellular signal-regulated kinases 1/2 and phospholipase Cgamma, in addition to inhibiting neurite outgrowth. In summary, our findings demonstrated that SAP may serve as a negative regulator of Trk receptor activation and downstream signaling.

Amino Acid Sequence↗
PubMed2003

Neurotrophin-3 down-regulates trkA mRNA, NGF high-affinity binding sites, and associated phenotype in adult DRG neurons.

Neurotrophin-3 (NT-3) binds to multiple trks, not only its initially identified receptor trkC. Recent studies in our laboratory show that NT-3 negatively regulates nociceptive phenotype associated with the trkA subpopulation. Due to the extensive overlap in trkA and trkC expression it is uncertain whether there is a direct influence of NT-3 on trkA in adult sensory neurons. Thus, the aim of this study was to examine whether NT-3 might alter trkA and associated neuronal phenotype outside of the trkC subpopulation. The effect of a seven-day intrathecal infusion of NT-3 on intact, uninjured adult rat dorsal root ganglion neurons was investigated. Serial sections were processed for receptor radioautography or in situ hybridization to identify and colocalize neurons expressing high-affinity nerve growth factor (NGF) binding sites, substance P (SP), trkC, or trkA mRNAs and to examine the influence of NT-3 on these populations. NT-3 does not appear to alter trkC expression, but evokes a notable reduction in trkA, high-affinity NGF binding sites, and SP levels. It is unlikely that signalling by trkC greatly influences this response because the down-regulation of SP occurs most notably in trkA neurons that lack trkC. Moreover, we have shown here that message levels of two trkA isoforms are differentially modulated by NT-3; infusion results in greater down-regulation of the noninsert containing isoform. These findings suggest a clinically relevant role for NT-3 as an antagonist to NGF, but also raise the caution that not just trkC-positive neurons are influenced following exposure to the neurotrophin.

Animals↗
PubMed1997

Interaction of NT-4 and BDNF with gp145trkb receptor: effect on cellular metabolism.

In the present study a silicon microphysiometer (Cytosensor) was applied in investigating interactions of gp145(trkb), a member of the tyrosine kinase receptor family, with different neurotrophic factors. NIH-3T3 cells transfected with gp145(trkb) receptors (NIH3T3/trkB cells) were utilized in the studies. Treatment with brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3) induced changes in the metabolic rate of NIH3T3/trkB cells. In contrast, no response was observed with nerve growth factor (NGF). The effects of NT-4 and BDNF on NIH3T3/trkB cells were receptor-specific in that they did not induce metabolic rate changes in wild type NIH3T3 cells or cells transfected with either gp140(trkb) (TrkA) or gp145(trkb) (TrkC) receptors. In contrast, NT-3 induced metabolic rate changes in cells transfected with each of the three different Trk receptors. The activity of NT-4 was significantly higher than that of BDNF. K252a, a protein kinase inhibitor, reduced the NT-4- and BDNF-induced response of the NIH3T3/trkB cells. This suggests that the NT-4 and BDNF-induced metabolic rate changes are associated with autophosphorylation of the tyrosine protein kinase residues. This hypothesis is further supported by results of western blot analysis. The results show that interactions of Trk receptors with neurotrophic factors result in metabolic changes in cells expressing the receptors.

3T3 Cells↗