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.

Neurotrophins play differential roles in short and long-term recognition memory.

The neurotrophin family of proteins are believed to mediate various forms of synaptic plasticity in the adult brain. Here we have assessed the roles of these proteins in object recognition memory in the rat, using icv infusions of function-blocking antibodies or the tyrosine kinase antagonist, tyrphostin AG879, to block Trk receptors. We report that tyrphostin AG879 impairs both short-term and long-term recognition memory, indicating a requirement for Trk receptor activation in both processes. The effect of inhibition of each of the neurotrophins with activity-blocking neutralising antibodies was also tested. Treatment with anti-BDNF, anti-NGF or anti-NT4 had no effect on short-term memory, but blocked long-term recognition memory. Treatment with anti-NT3 had no effect on either process. We also assessed changes in expression of neurotrophins and their respective receptors in the hippocampus, dentate gyrus and perirhinal cortex over a 24 h period following training in the object recognition task. We observed time-dependent changes in expression of the Trk receptors and their ligands in the dentate gyrus and perirhinal cortex. The data are consistent with a pivotal role for neurotrophic factors in the expression of recognition memory.

Interleukin 1-beta upregulates brain-derived neurotrophic factor, neurotrophin 3 and neuropilin 2 gene expression and NGF production in annulus cells.

The relationship between disc cells, nerves and pain production in the intervertebral disc is poorly understood. Neurotrophins, signaling molecules involved in the survival, differentiation and migration of neurons, and neurite outgrowth, are expressed in non-neuronal tissues including the disc. We hypothesized that three-dimensional exposure of human disc cells to the proinflammatory cytokine IL-1ß in vitro would elevate neurotrophin gene expression levels and production of nerve growth factor (NGF). Cells isolated from Thompson grade III and IV discs were cultured for 14 days under control conditions or with addition of 10(2) pM IL-1ß; mRNA was isolated and conditioned media assayed for NGF content. IL-1ß exposure in three-dimensional culture significantly increased expression of neurotrophin 3, brain-derived neurotrophic factor, and neuropilin 2 compared to controls. IL-1ß-exposed cells showed significantly increased NGF production compared to controls. Findings support our hypothesis, expand previous data concerning expression of neurotrophins, and provide the first documented expression of neurotrophin 3 and neuropilin 2. Our results have direct translational relevance, because they address the primary clinical issue of low back pain and open the possibility of novel analgesic therapies using specific small-molecular antagonists to neurotrophins.

Functional role of neurotrophin-3 in synapse regeneration by spiral ganglion neurons on inner hair cells after excitotoxic trauma in vitro.

Spiral ganglion neurons (SGNs) are postsynaptic to hair cells and project to the brainstem. The inner hair cell (IHC) to SGN synapse is susceptible to glutamate excitotoxicity and to acoustic trauma, with potentially adverse consequences to long-term SGN survival. We used a cochlear explant culture from P6 rat pups consisting of a portion of organ of Corti maintained intact with the corresponding portion of spiral ganglion to investigate excitotoxic damage to IHC-SGN synapses in vitro. The normal innervation pattern is preserved in vitro. Brief treatment with NMDA and kainate results in loss of IHC-SGN synapses and degeneration of the distal type 1 SGN peripheral axons, mimicking damage to SGN peripheral axons caused by excitotoxicity or noise in vivo. The number of IHC presynaptic ribbons is not significantly altered. Reinnervation of IHCs occurs and regenerating axons remain restricted to the IHC row. However, the number of postsynaptic densities (PSDs) does not fully recover and not all axons regrow to the IHCs. Addition of either neurotrophin-3 (NT-3) or BDNF increases axon growth and synaptogenesis. Selective blockade of endogenous NT-3 signaling with TrkC-IgG reduced regeneration of axons and PSDs, but TrkB-IgG, which blocks BDNF, has no such effect, indicating that endogenous NT-3 is necessary for SGN axon growth and synaptogenesis. Remarkably, TrkC-IgG reduced axon growth and synaptogenesis even in the presence of BDNF, indicating that endogenous NT-3 has a distinctive role, not mimicked by BDNF, in promoting SGN axon growth in the organ of Corti and synaptogenesis on IHCs.

GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity.

Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. We previously showed that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate, possibly by promoting neurite elongation via microtubule assembly. Here, we showed that glycogen synthase kinase-3beta (GSK-3beta) phosphorylated CRMP-2 at Thr-514 and inactivated it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3beta induced the formation of multiple axon-like neurites in hippocampal neurons. The expression of constitutively active GSK-3beta impaired neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracted the inhibitory effects of GSK-3beta, indicating that GSK-3beta regulates neuronal polarity through the phosphorylation of CRMP-2. Treatment of hippocampal neurons with neurotrophin-3 (NT-3) induced inactivation of GSK-3beta and dephosphorylation of CRMP-2. Knockdown of CRMP-2 inhibited NT-3-induced axon outgrowth. These results suggest that NT-3 decreases phosphorylated CRMP-2 and increases nonphosphorylated active CRMP-2, thereby promoting axon outgrowth.

The trophic role of oligodendrocytes in the basal forebrain.

Traditionally, the primary function of oligodendrocytes (OLGs) in the CNS has been considered to be myelination. Here, we investigated whether OLGs may play a trophic role, particularly during development. Neurotrophin expression was assessed in postnatal day 7 basal forebrain (BF) OLGs, using in situ hybridization and detection of myelin basic protein. Nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) mRNAs were revealed in OLGs in vivo and in culture. To determine whether OLGs support nearby neurons, we examined the influence of OLGs on BF cholinergic neurons. Neuronal function was enhanced by cocultured OLGs and OLG conditioned medium. Moreover, trophic effects of OLG conditioned medium were partially blocked by K252a, a trk tyrosine kinase inhibitor, and by neutralizing anti-BDNF or anti-NT-3 antisera, indicating that neurotrophins may mediate these effects, perhaps in concert with other signals. Our studies support a novel role for OLGs in providing local trophic support for neurons in the CNS.

Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways.

Cultured embryonic cortical progenitor cells will mimic the temporal differentiation pattern observed in vivo, producing neurons first and then glia. Here, we investigated the role of two endogenously produced growth factors, the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 (NT-3), in the early progenitor-to-neuron transition. Cultured cortical progenitors express BDNF and NT-3, as well as their receptors TrkB (tyrosine kinase receptor B) and TrkC. Inhibition of these endogenously expressed neurotrophins using function-blocking antibodies resulted in a marked decrease in the survival of cortical progenitors, accompanied by decreased proliferation and inhibition of neurogenesis. Inhibition of neurotrophin function also suppressed the downstream Trk receptor signaling pathways, PI3-kinase (phosphatidyl inositol-3-kinase) and MEK-ERK (MAP kinase kinase-extracellular signal-regulated kinase), indicating the presence of autocrine-paracrine neurotrophin:Trk receptor signaling in these cells. Moreover, specific inhibition of these two Trk signaling pathways led to distinct biological effects; inhibition of PI3-kinase decreased progenitor cell survival, whereas inhibition of MEK selectively blocked the generation of neurons, with no effects on survival or proliferation. Thus, neurotrophins made by cortical progenitor cells themselves signal through the TrkB and TrkC receptors to mediate cortical progenitor cell survival and neurogenesis via two distinct downstream signaling pathways.

Motoneuron-derived neurotrophin-3 is a survival factor for PAX2-expressing spinal interneurons.

Rat spinal cord interneurons undergo programmed cell death shortly after birth. We investigated here whether cell death of interneurons could be regulated by trophic factors produced by motoneurons, one of their main targets. To test this hypothesis, we studied the effect of the selective destruction of motoneurons on the survival of interneurons in organotypic cultures of embryonic rat spinal cords. Motoneurons were eliminated by an anti-p75(NTR)-specific immunotoxin (192 IgG-saporin). We then observed a decrease of 28% in the number of ventral spinal interneurons immunoreactive (IR) for the homeoprotein PAX2. This was correlated with an increase in the number of apoptotic nuclei in the same area. Because neurotrophin-3 (NT-3) is specifically produced by motoneurons and because interneurons express the NT-3 high-affinity receptor trkC, we examined the role of NT-3 in the survival of PAX2-IR interneurons. Addition of NT-3 to 192 IgG-saporin-treated explants rescued ventral PAX2-IR interneurons. Depletion of secreted NT-3 by anti-NT-3 antibodies induced 66% loss of ventral PAX2-IR interneurons. We conclude that motoneuron-derived NT-3 is a trophic factor for ventral PAX2-IR interneurons.

Neutralization of neutrophin-3 in the ventral tegmental area or nucleus accumbens differentially modulates cocaine-induced behavioral plasticity in rats.

These experiments were designed to assess the influence of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) in the mesoaccumbens dopamine system on the initiation of behavioral sensitization to cocaine. A neutralizing antibody for NT-3, BDNF or their vehicle was administered into the ventral tegmental area (VTA) or nucleus accumbens prior to each of four daily injections of 15 mg/kg cocaine. Behavioral sensitization was operationally defined as a significant increase in the behavioral response to cocaine relative to the first daily injection. Results indicated that the NT-3 antibody had differential effects when administered into the VTA or nucleus accumbens. Intra-VTA microinjection of anti-NT-3 resulted in enhanced sensitization to repeated cocaine injections in that the cocaine-induced behavioral response in the anti-NT-3 group was significantly greater than the vehicle group following the second and third daily injections of cocaine. Administration of anti-NT-3 into the nucleus accumbens increased the behavioral response to cocaine over all 4 days of cocaine administration, with no sensitization of this behavioral response. In contrast, pretreatment with anti-BDNF into the VTA or nucleus accumbens had no influence on the initiation of behavioral sensitization to cocaine. Taken together, these data indicate that neutralization of NT-3 in the VTA enhances cocaine-induced behavioral sensitization, while administration of the NT-3 antibody into the nucleus accumbens increases the hyperactive behavioral response induced by cocaine but impairs the further development of behavioral sensitization.

Sequential interactions of fibroblast growth factor-2, brain-derived neurotrophic factor, neurotrophin-3, and their receptors define critical periods in the development of cochlear ganglion cells.

We studied the interactions of neurotrophin-3 (NT3) with brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF-2), and their effects on tyrosine kinase C (TrkC) expression during cochlear ganglion development. Otocysts were explanted from white leghorn chicken embryos at stages when the neuronal precursors normally start to migrate. Cultures were fed with various combinations of NT3, BDNF, and FGF-2. NT3 appeared to have a greater effect on neurite outgrowth than on migration and was enhanced by BDNF. The results from in situ hybridization and immunostaining for TrkC receptor revealed up-regulation of the mRNA and protein by combining NT-3 and BDNF. NT-3 combined with FGF-2 produced down-regulation of receptor. Neutralizing antibody to NT3 had an inhibitory effect on neuronal development, suggesting that endogenous NT3 is normally active during the period examined. The findings suggest an interactive role of NT3 in early neuronal development. The trophic synergism of NT3 and BDNF may result from up-regulation of TrkC. This hypothesis is consistent with immunostaining in the embryonic basilar papilla, which localized TrkC to the initial axonal invasion sites. While the growth factors each produce particular trophic effects, the interactions of these factors define a critical sequence of developmental events based on modulation of receptor expression.

Hyperbaric oxygen treatment decreases post-ischemic neurotrophin-3 mRNA down-regulation in the rat hippocampus.

The therapeutic effect of hyperbaric oxygen (HBO) on ischemic injury was investigated using in situ hybridization to detect the mRNA expression of neurotrophin-3 (NT-3), which is thought to play a crucial role in protecting against neuronal death induced by brain ischemia. The rats under investigation were subjected to 10 min transient forebrain ischemia, and subsequently exposed to HBO (100% oxygen, 2.5 atm absolute) for 2 h. Levels of NT-3 mRNA in the CA1, CA2 and CA3 regions, and the dentate gyrus of the hippocampus were measured after various reperfusion periods. Neuronal death in the hippocampal CA1 region was also measured by Nissl staining, seven days post ischemia. The results demonstrated that HBO treatment significantly reduced the ischemia-induced down-regulation of the NT-3 mRNA level at 4 h post ischemia, and significantly increased cell survival 7 days after reperfusion. The findings suggest that an HBO treatment maintaining the NT-3 mRNA level in the hippocampus can be beneficial to the ischemic brain within a certain time frame.

An activity-dependent neurotrophin-3 autocrine loop regulates the phenotype of developing hippocampal pyramidal neurons before target contact.

Neurotrophin-3 (NT-3), its cognate receptor trkC, and voltage-gated calcium channels are coexpressed by embryonic pyramidal neurons before target contact, but their functions at this stage of development are still unclear. We show here that, in vitro, anti-NT-3 and anti-trkC antibodies blocked the increase, and NT-3 reversed the decrease in the number of calbindin-D(28k)-positive pyramidal neurons induced by, respectively, calcium channel activations and blockades. Similar results were obtained with single-neuron microcultures. In addition, voltage-gated calcium channel inhibition downregulates the extracellular levels of NT-3 in high-density cultures. Moreover, electrophysiological experiments in single-cell cultures reveal a tetrodotoxin-sensitive spontaneous electrical activity allowing voltage-gated calcium channel activation. The mouse NT-3 (-/-) mutation decreases by 40% the number of developing calbindin-D(28k)-positive pyramidal neurons, without affecting neuronal survival, both in vitro and in vivo. Thus, present results strongly support that an activity-dependent autocrine NT-3 loop provides a local, intrinsic mechanism by which, before target contact, hippocampal pyramidal-like neurons may regulate their own differentiation, a role that may be important during early CNS differentiation or after adult target disruption.

Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.

Neurotrophin-3 (NT-3) promotes enteric neuronal development in vitro; nevertheless, an enteric nervous system (ENS) is present in mice lacking NT-3 or TrkC. We thus analyzed the physiological significance of NT-3 in ENS development. Subsets of neurons developing in vitro in response to NT-3 became NT-3 dependent; NT-3 withdrawal led to apoptosis, selectively in TrkC-expressing neurons. Antibodies to NT-3, which blocked the developmental response of enteric crest-derived cells to exogenous NT-3, did not inhibit neuronal development in cultures of isolated crest-derived cells but did so in mixed cultures of crest- and non-neural crest-derived cells; therefore, the endogenous NT-3 that supports enteric neuronal development is probably obtained from noncrest-derived mesenchymal cells. In mature animals, retrograde transport of (125)I-NT-3, injected into the mucosa, labeled neurons in ganglia of the submucosal but not myenteric plexus; injections of (125)I-NT-3 into myenteric ganglia, the tertiary plexus, and muscle, labeled neurons in underlying submucosal and distant myenteric ganglia. The labeling pattern suggests that NT-3-dependent submucosal neurons may be intrinsic primary afferent and/or secretomotor, whereas NT-3-dependent myenteric neurons innervate other myenteric ganglia and/or the longitudinal muscle. Myenteric neurons were increased in number and size in transgenic mice that overexpress NT-3 directed to myenteric ganglia by the promoter for dopamine beta-hydroxylase. The numbers of neurons were regionally reduced in both plexuses in mice lacking NT-3 or TrkC. A neuropoietic cytokine (CNTF) interacted with NT-3 in vitro, and if applied sequentially, compensated for NT-3 withdrawal. These observations indicate that NT-3 is required for the normal development of the ENS.

Endogenous brain-derived neurotrophic factor and neurotrophin-3 antagonistically regulate survival of axotomized corticospinal neurons in vivo.

Neuronal growth factors regulate the survival of neurons by their survival and death-promoting activity on distinct populations of neurons. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) promote neuronal survival via tyrosine kinase (Trk) receptors, whereas NGF and BDNF can also induce apoptosis in developing neurons through p75(NTR) receptors in the absence of their respective Trk receptors. Using mutant mice and inactivation of neurotrophins and their receptors with antibodies in rats, we show that endogenous NT-3 induces death of adult BDNF-dependent, axotomized corticospinal neurons (CSNs). When NT-3 is neutralized, the neurons survive even without BDNF, suggesting complete antagonism. Whereas virtually all unlesioned and axotomized CSNs express both trkB and trkC mRNA, p75 is barely detectable in unlesioned CSNs but strongly upregulated in axotomized CSNs by day 3 after lesion, the time point when cell death occurs. Blocking either cortical TrkC or p75(NTR) receptors alone prevents death, indicating that the opposing actions of NT-3 and BDNF require their respective Trk receptors, but induction of death depends on p75(NTR) cosignaling. The results show that neuronal survival can be regulated antagonistically by neurotrophins and that neurotrophins can induce neuronal death in the adult mammalian CNS. We further present evidence that signaling of tyrosine kinase receptors of the trk family can be crucially involved in the promotion of neuronal death in vivo.

Neurotrophin-3 mediates the autocrine survival of the catecholaminergic CAD CNS neuronal cell line.

The mechanisms for neuronal survival in the CNS are not well understood, but are likely to be complex due to possible autocrine and redundant neurotrophic support. Most studies have focused on the nerve growth factor (NGF)/TrkA pathway in peripheral neurons, and little is known regarding the other neurotrophins, particularly neurotrophin-3 (NT3)/TrkC. Progress has also been hampered by the paucity of homogenous and accessible CNS neuronal experimental models. We now report that the novel catecholaminergic CNS cell line, CAD, is capable of autocrine survival mediated by NT3. The CAD cell is of CNS neuronal origin and can survive and morphologically differentiate in the absence of exogenously provided trophic factors. However, neutralizing reagents against NT3 (the neutralizing TrkC-IgG fusion protein and anti-NT3 antibodies), but not those that block the other neurotrophins, inhibited survival of differentiating CAD cells. Moreover, Trk phosphorylation was detected in CAD cells and its inhibition by K252a was correlated with K252a-induced apoptosis. Finally, endogenous NT3 was detectable in CAD cell extracts by a specific ELISA assay. Thus, CAD cells possess an autocrine survival capability mediated by NT3, and may provide a valuable model system for studying the signaling pathways that mediate the actions of this little understood neurotrophin.

Neurotrophin-3 promotes the cholinergic differentiation of sympathetic neurons.

Neurotrophins influence the epigenetic shaping of the vertebrate nervous system by regulating neuronal numbers during development and synaptic plasticity. Here we attempt to determine whether these growth factors can also regulate neurotransmitter plasticity. As a model system we used the selection between noradrenergic and cholinergic neurotransmission by paravertebral sympathetic neurons. Developing sympathetic neurons express the neurotrophin receptors TrkA and TrkC, two highly related receptor tyrosine kinases. Whereas the TrkA ligand nerve growth factor (NGF) has long been known to regulate both the survival and the expression of noradrenergic traits in sympathetic neurons, the role of TrkC and of its ligand neurotrophin-3 (NT3) has remained unclear. We found that TrkC expression in the avian sympathetic chain overlaps substantially with that of choline acetyltransferase. In sympathetic chain explants, transcripts of the cholinergic marker genes choline acetyltransferase and vasoactive intestinal polypeptide were strongly enriched in the presence of NT3 compared with NGF, whereas the noradrenergic markers tyrosine hydroxylase and norepinephrine transporter were reduced. The transcription factor chicken achaete scute homolog 1 was coexpressed with cholinergic markers. The effects of NT3 are reversed and antagonized by NGF. They are independent of neuronal survival and developmentally regulated. These results suggest a role for NT3 as a differentiation factor for cholinergic neurons and establish a link between neurotrophins and neurotransmitter plasticity.

Effects of endogenous neurotrophins on sympathetic sprouting in the dorsal root ganglia and allodynia following spinal nerve injury.

Peripheral nerve injury is often complicated by a chronic pain syndrome that is difficult to treat. In animal models of peripheral nerve injury, sympathetic nerve terminals in the dorsal root ganglia (DRG) sprout to form baskets around large diameter neurons, an anatomical change that has been implicated in the induction of neuropathic pain. In the present study, we have investigated whether neurotrophins derived from peripheral sources play any roles in sympathetic sprouting and neuropathic pain in a rat model of peripheral nerve injury. After transection of the left lumbar (L) 5 spinal nerve, antisera specific to neurotrophins were injected intraperitoneally twice a week for 2 weeks. The foot withdrawal response to von Frey hairs was examined on days 1, 3, 7, 10, and 14 postlesion. After completion of behavioral tests, sympathetic sprouting in DRG was examined by tyrosine hydroxylase (TH) immunohistochemistry. The number of TH-immunoreactive (ir) fibers and baskets around large neurons within the lesioned DRG was dramatically increased in the rats treated with control normal sheep serum. Antisera specific to nerve growth factor (NGF), neurotrophin-3 (NT3), and brain-derived neurotrophic factor (BDNF) significantly reduced the sympathetic sprouting and the formation of baskets. L5 spinal nerve lesion induced a significant increase in foot withdrawal responses to von Frey hair stimuli, which was attenuated by treatment of antisera to neurotrophins with a different time sequential. The effect of BDNF antiserum occurred earlier and lasted longer than those of NGF and NT3 antisera. These results implicate that peripherally derived neurotrophins are involved in the induction of sympathetic sprouting and neuropathic pain following peripheral nerve injury.

Neurotrophins from dorsal root ganglia trigger allodynia after spinal nerve injury in rats.

Injury to peripheral nerves often results in chronic pain which is difficult to relieve. The mechanism underlying the pain syndrome remains largely unknown. In previous studies we showed that neurotrophins are up-regulated in satellite cells around sensory neurons following sciatic nerve lesion. In the present study, we have examined whether the neurotrophins in the dorsal root ganglia play any role in allodynia after nerve injury. Antibodies to different neurotrophins, directly delivered to injured dorsal root ganglia, significantly reduced (with different time sequences) the percentage of foot withdrawal responses evoked by von Frey hairs. The antibodies to nerve growth factor acted during the early phase but antibodies to neurotrophin-3 and brain-derived neurotrophic factor were effective during the later phase. Exogenous nerve growth factor or brain-derived neurotrophic factor, but not neurotrophin-3, directly delivered to intact dorsal root ganglia, trigger a persistent mechanical allodynia. Our results showed that neurotrophins within the dorsal root ganglia after peripheral nerve lesion are involved in the generation of allodynia at different stages. These studies provide the first evidence that ganglia-derived neurotrophins are a source of nociceptive stimuli for neuropathic pain after peripheral nerve injury.