[Progress in the role of neurotrophin-3 and its receptors in the development of cochlear spiral ganglion in rats].

Neurotrophin-3 (NT-3) attracted increasing attention about NTFs researches in recent years. But the mechanism of promoting the development of neurons and neurite extension is not clear. Recombinant human NT-3 or NT-3 gene is commonly used in the treatment of peripheral and central neurons system damage. When rats are born, the cochlear is not mature yet. It is a suitable experimental animal for studying the morphological and functional development of the peripheral auditory pathway. It was found that NT-3 could promote the survival, growth, division and extension of the cochlear neurons in rats. To make clear the role of NT-3 in the development of spiral ganglion in the rat cochlear will be of significance for the treatment of nervous hearing loss by NT-3 in the future.

Neurotrophin-3 modulates breast cancer cells and the microenvironment to promote the growth of breast cancer brain metastasis.

Metastasis, which remains incompletely characterized at the molecular and biochemical levels, is a highly specific process. Despite the ability of disseminated cancer cells to intravasate into distant tissues, it has been long recognized that only a limited subset of target organs develop clinically overt metastases. Therefore, subsequent adaptation of disseminated cancer cells to foreign tissue microenvironment determines the metastatic latency and tissue tropism of these cells. As a result, studying interactions between the disseminated cancer cells and the adjacent stromal cells will provide a better understanding of what constitutes a favorable or unfavorable microenvironment for disseminated cancer cells in a tissue-specific manner. Previously, we reported a protein signature of brain metastasis showing increased ability of brain metastatic breast cancer cells to counteract oxidative stress. In this study, we showed that another protein from the brain metastatic protein signature, neurotrophin-3 (NT-3), has a dual function of regulating the metastatic growth of metastatic breast cancer cells and reducing the activation of immune response in the brain. More importantly, increased NT-3 secretion in metastatic breast cancer cells results in a reversion of mesenchymal-like (EMT) state to epithelial-like (MET) state and vice versa. Ectopic expression of NT-3 in EMT-like breast cancer cells reduces their migratory ability and increases the expression of HER2 (human epidermal growth factor receptor 2) and E-cadherin at the cell-cell junction. In addition, both endogenous and ectopic expression of NT-3 reduced the number of fully activated cytotoxic microglia. In summary, NT-3 appears to promote growth of metastatic breast cancer cells in the brain by facilitating the re-epithelialization of metastatic breast cancer cells and downmodulating the cytotoxic response of microglia. Most importantly, our results provide new insights into the latency and development of central nervous system macrometastases in patients with HER2-positive breast tumors and provide mechanistic rationale to target HER2 signaling for HER2-positive breast cancer brain metastasis.

TrkC signaling is activated in adenoid cystic carcinoma and requires NT-3 to stimulate invasive behavior.

Treatment options for adenoid cystic carcinoma (ACC) of the salivary gland, a slowly growing tumor with propensity for neuroinvasion and late recurrence, are limited to surgery and radiotherapy. Based on expression analysis performed on clinical specimens of salivary cancers, we identified in ACC expression of the neurotrophin-3 receptor TrkC/NTRK3, neural crest marker SOX10, and other neurologic genes. Here, we characterize TrkC as a novel ACC marker, which was highly expressed in 17 out of 18 ACC primary-tumor specimens, but not in mucoepidermoid salivary carcinomas or head and neck squamous cell carcinoma. Expression of the TrkC ligand NT-3 and Tyr-phosphorylation of TrkC detected in our study suggested the existence of an autocrine signaling loop in ACC with potential therapeutic significance. NT-3 stimulation of U2OS cells with ectopic TrkC expression triggered TrkC phosphorylation and resulted in Ras, Erk 1/2 and Akt activation, as well as VEGFR1 phosphorylation. Without NT-3, TrkC remained unphosphorylated, stimulated accumulation of phospho-p53 and had opposite effects on p-Akt and p-Erk 1/2. NT-3 promoted motility, migration, invasion, soft-agar colony growth and cytoskeleton restructuring in TrkC-expressing U2OS cells. Immunohistochemical analysis demonstrated that TrkC-positive ACC specimens also show high expression of Bcl2, a Trk target regulated via Erk 1/2, in agreement with activation of the TrkC pathway in real tumors. In normal salivary gland tissue, both TrkC and Bcl2 were expressed in myoepithelial cells, suggesting a principal role for this cell lineage in the ACC origin and progression. Sub-micromolar concentrations of a novel potent Trk inhibitor AZD7451 completely blocked TrkC activation and associated tumorigenic behaviors. Pre-clinical studies on ACC tumors engrafted in mice showed efficacy and low toxicity of AZD7451, validating our in vitro data and stimulating more research into its clinical application. In summary, we describe in ACC a previously unrecognized pro-survival neurotrophin signaling pathway and link it with cancer progression.

Gene expression in skin, muscle, and dorsal root ganglion after plantar incision in the rat.

BACKGROUND: Treating postoperative pain remains a significant challenge for perioperative medicine. Recent studies have shown that nerve growth factor is up-regulated and contributes to incisional pain. To date, few studies have examined expression of other neurotrophin-related mediators that may contribute to the development and/or maintenance of incisional pain. METHODS: Male Sprague-Dawley rats underwent a plantar incision, and pain behaviors were examined (n = 6). In a separate group of rats, expression of neurotrophic factors were studied. At various times after incision (n = 4) or sham surgery (n = 4), the skin, muscle, and dorsal root ganglia were harvested and total RNA isolated. Real-time reverse transcription polymerase chain reaction was performed and the fold change in gene expression was analyzed using significance analysis of microarrays. RESULTS: Several genes were changed (P < 0.05) as early as 1 h after incision. Expression of artemin and nerve growth factor were increased in both incised skin and muscle. Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-5 were all down-regulated in the skin but up-regulated in the muscle 48 h after incision. Few genes changed in the dorsal root ganglion. Most changes in expression occurred in the first 48 h after incision, a timeframe when pain behavior was the greatest. CONCLUSION: Surgical incision is associated with pain-related gene expression changes in skin, muscle, and, to a lesser extent, dorsal root ganglion. The gene expression profile provides clues as to mediators that are involved in peripheral sensitization and pain transmission after surgical incision and also suggest mechanisms for resolution of postoperative pain when more persistent pain syndromes like neuropathic pain continue.

Insulin-like growth factor binding protein-2 and neurotrophin 3 synergize together to promote the expansion of hematopoietic cells ex vivo.

Co-culture of Umbilical Cord Blood (UCB) CD34+ cells with irradiated Mesenchymal Stem Cells (MSCs) without contact increase the expansion of Hematopoietic Progenitor Cells (HPC). Neurotrophin-3 (NT-3) and insulin-like growth factor binding protein-2 (IGFBP-2) are two factors whose expressions were significantly elevated in conditioned media derived from irradiated MSCs. To determine whether these factors are partly responsible for the growth promoting potential of MSCs, we investigated their impact on the growth and differentiation of UCB-CD34+ cells. Addition of either factor alone had little impact on cell growth, however both factors synergized together to increase the expansion of total nucleated cells, erythroids, megakaryocytes (Mk) and CD34+ cells. However, in contrast to MSCs they failed to significantly improve the expansion of hematopoietic progenitors. Consistent with the impact of these factors on hematopoietic cells, both synergized to activate ERK1/2 and AKT in primary human UCB cells. In conclusion, the study demonstrates for the first time that a neurotrophin factor can synergize with IGFBP-2 to promote hematopoietic cell expansion.

Neurotrophin gene therapy for sustained neural preservation after deafness.

The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the residual spiral ganglion neurons. These neurons, however, undergo progressive degeneration after hearing loss, marked initially by peripheral fibre retraction and ultimately culminating in cell death. This research aims to use gene therapy techniques to both hold and reverse this degeneration by providing a sustained and localised source of neurotrophins to the deafened cochlea. Adenoviral vectors containing green fluorescent protein, with or without neurotrophin-3 and brain derived neurotrophic factor, were injected into the lower basal turn of scala media of guinea pigs ototoxically deafened one week prior to intervention. This single injection resulted in localised and sustained gene expression, principally in the supporting cells within the organ of Corti. Guinea pigs treated with adenoviral neurotrophin-gene therapy had greater neuronal survival compared to contralateral non-treated cochleae when examined at 7 and 11 weeks post injection. Moreover; there was evidence of directed peripheral fibre regrowth towards cells expressing neurotrophin genes after both treatment periods. These data suggest that neurotrophin-gene therapy can provide sustained protection of spiral ganglion neurons and peripheral fibres after hearing loss.

Involvement of spinal neurotrophin-3 in electroacupuncture analgesia and inhibition of spinal glial activation in rat model of monoarthritis.

UNLABELLED: Although electroacupuncture (EA) has been proven to effectively relieve pain associated with arthritis, the underlying mechanism of EA analgesia requires further investigation. Here, the involvement of spinal neurotrophin-3 (NT-3) in EA's analgesic effects on complete Freund's adjuvant (CFA)-induced inflammatory pain was examined. The present study demonstrated that: 1) repeated EA stimulation of ipsilateral GB30 and GB34 acupoints remarkably suppressed CFA-induced hyperalgesia; 2) EA treatment markedly enhanced the upregulation of spinal NT-3 mRNA and protein levels following CFA injection; 3) antisense oligodeoxynucleotides (ODN) specifically against NT-3 intrathecally administered during EA treatment for 7 days significantly attenuated the EA analgesia; and 4) the suppressed expression of spinal GFAP (astrocytic marker), OX-42 (microglial marker) as well as proinflammatory cytokines, interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α by EA treatment was significantly attenuated following NT-3 antisense ODN delivery. These results suggested that endogenous NT-3 may be involved in the analgesic effect of EA on inflammatory pain in rats, mediated through the inhibition of spinal glial activity as well as proinflammatory cytokine production. PERSPECTIVE: The present study may initiate a discussion on the possible roles of NT-3/glia/cytokines in the therapeutic effects of acupuncture and provide insight on the mechanism underlie the analgesic effects of acupuncture on pain associated with arthritis.

Functionally reduced sensorimotor connections form with normal specificity despite abnormal muscle spindle development: the role of spindle-derived neurotrophin 3.

The mechanisms controlling the formation of synaptic connections between muscle spindle afferents and spinal motor neurons are believed to be regulated by factors originating from muscle spindles. Here, we find that the connections form with appropriate specificity in mice with abnormal spindle development caused by the conditional elimination of the neuregulin 1 receptor ErbB2 from muscle precursors. However, despite a modest ( approximately 30%) decrease in the number of afferent terminals on motor neuron somata, the amplitude of afferent-evoked synaptic potentials recorded in motor neurons was reduced by approximately 80%, suggesting that many of the connections that form are functionally silent. The selective elimination of neurotrophin 3 (NT3) from muscle spindles had no effect on the amplitude of afferent-evoked ventral root potentials until the second postnatal week, revealing a late role for spindle-derived NT3 in the functional maintenance of the connections. These findings indicate that spindle-derived factors regulate the strength of the connections but not their initial formation or their specificity.

Combination of microsurgery and gene therapy for spinal dorsal root injury repair.

Brachial plexus injury is frequent after traffic accident in adults or shoulder dystocia in newborns. Whereas surgery can restore arm movements, therapeutic options are missing for sensory defects. Dorsal root (DR) ganglion neurons convey sensory information to the central nervous system (CNS) through a peripheral and a central axon. Central axons severed through DR section or avulsion during brachial plexus injury inefficiently regenerate and do not reenter the spinal cord. We show that a combination of microsurgery and gene therapy circumvented the functional barrier to axonal regrowth at the peripheral and CNS interface. After cervical DR section in rats, microsurgery restored anatomical continuity through a nerve graft that laterally connected the injured DR to an intact DR. Gene transfer to cells in the nerve graft induced the local release of neurotrophin-3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) and stimulated axonal regrowth. Central DR ganglion axons efficiently regenerated and invaded appropriate areas of the spinal cord dorsal horn, leading to partial recovery of nociception and proprioception. Microsurgery created conditions for functional restoration of DR ganglion central axons, which were improved in combination with gene therapy. This combination treatment provides means to reduce disability due to somatosensory defects after brachial plexus injury.

[Influence of astrocyte-conditioned medium on the formation of synapses in neural stem cells: the role of neurotrophin proteins].

OBJECTIVE: To discuss whether neurotrophin proteins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neural growth factor (NGF), in the astrocyte-conditioned medium (ACM) are involved in the synapse formation in neural stem cells (NSCs). METHODS: (1) Cells derived from a pheochromocytoma of the rat adrenal medulla of the line PC12 were induced by amyloid-beta protein (Abeta)1-40 for 0, 4, 6, 12, and 24 h respectively. Then part of these PC12 cells underwent flow cytometry to examine the apoptotic rates. Different cells were added into Falcon Cell Culture Insert: Group A containing astrocytes isolated from Wistar rat, Group B with PC12 cells and astrocytes, Groups C1-C5 containing astrocytes and PC12 cells induced by Abeta(1-40) for 0, 4, 6, 12, and 24 h respectively, Group D1-5 with PC12 cells induced by Abeta(1-40) for 0, 4, 6, 12, and 24 h respectively, and Group E containing astrocytes induced by Abeta(1-40) for 6 h. Flow cytometry was used to detect the apoptotic rates of different groups. Double-antibody sandwich ELISA was used to detect the levels of BDNF, NT-3, and NGF. (2) The different kinds of the astrocyte-conditioned medium as described above were mixed with DMEM/F12 medium according to the proportion of 1:3 and then divided into 13 groups: Group I (Group A + NSCs), Group II (Group B + NSCs), Group III-VII (Groups C1-C5 + NSCs), Group VIII (NSCs without ASM), Group IX-XIII (Groups D1-D5 + Mscs), and Group XIV (Group E + NSCs). The expression of synaptophysin and growth-associated protein-43 (GAP-43 protein) were detected by co-focal laser scanning microscopy. The number of mature synapse was observed by transmission electron microscope(TEM). RESULTS: Flow cytometry showed that the apoptotic rates of the PC12 cells were low 0, 2, and 4 h after Abeta(1-40) induction, with the peak 6 h after induction (P < 0.05). The BDNF total protein level in the ACM of Group C3 was the highest (A = 1.53 +/- 0.25) (P < 0.05). The expression levels of synaptophysin (A = 33.39 +/- 2.71) and GAP-43 (A = 49.18 +/- 6.45), and the mature synapse number of NSCs (4.70 +/- 0.52 synapse/field of vision) of Group V were the highest in comparison with the other groups (all P < 0.05). CONCLUSION: After incubation of astrocytes with Abeta(1-40)-induced PC12 cells(Abeta-PC12), the ACM induces the synapse formation in the NSCs. The BDNF in the ACM is probably involved in this process.

Mash1 and neurogenin 2 enhance survival and differentiation of neural precursor cells after transplantation to rat brains via distinct modes of action.

Neural precursor cells (NPCs) are regarded as a promising source of donor cells in transplantation-based therapies for neurodegenerative disorders. However, poor survival and limited neuronal differentiation of the transplanted NPCs remain critical limitations for developing therapeutic strategies. In this study, we investigated the effects of the proneural basic helix-loop-helix (bHLH) transcription factors Mash1 and Neurogenin 2 (Ngn2) in neuronal differentiation and survival of NPCs. Induction of Mash1 or Ngn2 expression strikingly enhanced neuronal differentiation of cultured NPCs in vitro. Ngn2-transduced NPCs underwent a rapid cell cycle arrest, which often accompanies differentiation. In contrast, cells continuously expanded upon Mash1 expression during NPC differentiation. Notably, sonic hedgehog (SHH) was upregulated by Mash1 and mediated the proliferative and survival effects of Mash1 on NPCs. Upon transplantation into adult rat brains, Mash1-expressing NPCs yielded large grafts enriched with neurons compared to control LacZ-transduced NPCs. Interestingly, enhancements in neuronal yield, as well as in donor cell survival, were also achieved by transplanting Ngn2-transduced NPCs. We show that a differentiation stage- and cell density-dependent survival effect of Ngn2 involves neurotrophin3 (NT3)/TrkC-mediated signaling. Together, these findings suggest potential benefits of bHLH gene manipulation to develop successful transplantation strategies for brain disorders.

Differential up-regulation of neurotrophin receptors and functional activity of neurotrophins on peripheral blood eosinophils of patients with allergic rhinitis, atopic dermatitis and nonatopic subjects.

BACKGROUND: Recent studies suggest that neurotrophins have a pivotal role in neuroimmune interactions. Indeed, in contrast to nonatopic subjects (NA), neurotrophins have been shown to be increased in atopic diseases such as allergic rhinitis (AR) and atopic dermatitis (AD). AIM: The aim of the study was to assess the functional role of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and -4 and the expression of pan-neurotrophin receptor (p75(NTR)) and tyrosine kinase (trk)A, -B and -C on peripheral blood eosinophils in AR, AD and NA. METHODS: Peripheral blood eosinophils of patients with AR, AD and NA were purified by CD16 negative selection (purity>98%). Neurotrophin receptor expression was analysed by FACS analysis. Apoptosis test (FACS analysis) and chemotactic index (modified Boyden chamber assay) were assessed after stimulation with BDNF, NT-3/-4 and NGF. RESULTS: The expression of trkA-C and p75(NTR) was significantly higher in AD>AR>NA (P<0.05-0.001). Apoptosis was significantly inhibited by BDNF, NGF, NT-3 in AD (P<0.05-0.001), by NT-3/-4 and NGF in AR (P<0.05-0.01) and by NT-3 (P<0.05-0.01) in NA eosinophils. Chemotaxis was significantly induced by BDNF and NT-3/4 (P<0.01-0.001) in AD peripheral blood eosinophils. CONCLUSION: Neurotrophin receptor expression and neurotrophin functional activity was greatest in AD>AR>NA. AD eosinophils are pre-activated and may therefore better respond to neurotrophins. With this study, we provide new pathophysiologic insights into atopic diseases with a functional role of neurotrophins in peripheral blood eosinophils in AD and AR.

Increased chondroitin sulfate proteoglycan expression in denervated brainstem targets following spinal cord injury creates a barrier to axonal regeneration overcome by chondroitinase ABC and neurotrophin-3.

Increased chondroitin sulfate proteoglycan (CSPG) expression in the vicinity of a spinal cord injury (SCI) is a primary participant in axonal regeneration failure. However, the presence of similar increases of CSPG expression in denervated synaptic targets well away from the primary lesion and the subsequent impact on regenerating axons attempting to approach deafferented neurons have not been studied. Constitutively expressed CSPGs within the extracellular matrix and perineuronal nets of the adult rat dorsal column nuclei (DCN) were characterized using real-time PCR, Western blot analysis and immunohistochemistry. We show for the first time that by 2 days and through 3 weeks following SCI, the levels of NG2, neurocan and brevican associated with reactive glia throughout the DCN were dramatically increased throughout the DCN despite being well beyond areas of trauma-induced blood brain barrier breakdown. Importantly, regenerating axons from adult sensory neurons microtransplanted 2 weeks following SCI between the injury site and the DCN were able to regenerate rapidly within white matter (as shown previously by Davies et al. [Davies, S.J., Goucher, D.R., Doller, C., Silver, J., 1999. Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord. J. Neurosci. 19, 5810-5822]) but were unable to enter the denervated DCN. Application of chondroitinase ABC or neurotrophin-3-expressing lentivirus in the DCN partially overcame this inhibition. When the treatments were combined, entrance by regenerating axons into the DCN was significantly augmented. These results demonstrate both an additional challenge and potential treatment strategy for successful functional pathway reconstruction after SCI.

Regulation of sympathetic neuron differentiation by endogenous nerve growth factor and neurotrophin-3.

Nerve growth factor (NGF) and neurotrophin-3 (NT3) play distinctive roles in sympathetic axon growth and target field innervation and are required for sympathetic neuron survival in vivo. To ascertain if these neurotrophins selectively regulate the expression of genes that determine the functional characteristics of differentiated sympathetic neurons, we measured the mRNA levels for several such genes in the superior cervical ganglion of NGF(-/-), NT3(-/-) and wild type mouse embryos at a stage before excessive neuronal loss occurs in the absence of these neurotrophins. Despite the extensively documented ability of NGF to regulate the noradrenergic phenotype of sympathetic neurons, we found that tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) mRNA levels were normal in NGF(-/-) embryos, but significantly reduced in NT3(-/-) embryos. In contrast, the beta2 nicotinic acetylcholine receptor and PACAP receptor 1 mRNA levels were normal in NT3(-/-) embryos, but significantly reduced in NGF(-/-) embryos. Studies of mice lacking neurotrophin receptors suggested that the effects of NGF on gene expression require TrkA whereas those of NT3 require TrkA and p75(NTR). These findings demonstrate that endogenous NGF and NT3 have distinctive and separate effects on gene expression in early sympathetic neurons and that these selective effects on gene expression require a different combination of neurotrophin receptors.

[Skin changes in amyotrophic lateral sclerosis].

It has been repeatedly noted, but never as yet fully explained, that patients with amyotrophic lateral sclerosis (ALS) do not develop bedsores even at the terminal stage. Furthermore, the skin of ALS patients feels supple, like tanned leather, and loses elasticity. When the skin is stretched, it returns only sluggishly to its original position. We termed this property of skin "delayed return phenomenon (DRP)"; it is usually seen more than 2.5 years after the onset of symptoms. Although it is thought that a phenomena such as DRP and the absence of bedsores are characteristic of this disease, little attention has been paid to these unique features in ALS patients. In this review we summarize recent developments in research on skin from ALS patients. From our own works cited in this review it is clear that not only the motor neuron but also the skin is affected in ALS, and that abnormalities of collagen, glycosaminoglycans, vascular endotherial growth factor (VEGF) and neurotrophic factors like ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3) and insulin-like growth factor-1 (IGF-1) do occur in the skin of ALS. Examination of the skin in patients with ALS would be easy to carry out as an additional examination. Further analysis of the complex skin abnormalities will be useful in elucidating the basic pathological mechanism of ALS.

Post-injury regeneration in rat sciatic nerve facilitated by neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.

Amniotic fluid mesenchymal stem cells have the ability to secrete neurotrophic factors that are able to promote neuron survival in vitro. The purpose of this study was to evaluate the effects of neurotrophic factors secreted by rat amniotic fluid mesenchymal stem cells on regeneration of sciatic nerve after crush injury. Fifty Sprague-Dawley rats weighing 250-300 g were used. The left sciatic nerve was crushed with a vessel clamp. Rat amniotic fluid mesenchymal stem cells embedded in fibrin glue were delivered to the injured nerve. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to detect neurotrophic factors secreted by the amniotic fluid mesenchymal stem cells. Nerve regeneration was assessed by motor function, electrophysiology, histology, and immunocytochemistry studies. Positive CD29/44, and negative CD11b/45, as well as high levels of expression of brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor, ciliary neurotrophic factor (CNTF), nerve growth factor, and neurotrophin-3 (NT-3) were demonstrated in amniotic fluid mesenchymal stem cells. Motor function recovery, the compound muscle action potential, and nerve conduction latency showed significant improvement in rats treated with amniotic fluid mesenchymal stem cells. ELISA measurement in retrieved nerves displayed statistically significant elevation of CNTF and NT-3. The immunocytochemical studies demonstrated positive staining for NT-3 and CNTF in transplanted cells. The histology and immunocytochemistry studies revealed less fibrosis and a high level of expression of S-100 and glial fibrillary acid protein at the crush site. Rat amniotic fluid mesenchymal stem cells may facilitate regeneration in the sciatic nerve after crush injury. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.

Postsynaptic secretion of BDNF and NT-3 from hippocampal neurons depends on calcium calmodulin kinase II signaling and proceeds via delayed fusion pore opening.

The mammalian neurotrophins (NTs) NGF, BDNF, NT-3, and NT-4 constitute a family of secreted neuronal growth factors. In addition, NTs are implicated in several forms of activity-dependent synaptic plasticity. Although synaptic secretion of NTs has been described, the intracellular signaling cascades that regulate synaptic secretion of NTs are far from being understood. Analysis of NT secretion at the subcellular level is thus required to resolve the role of presynaptic and postsynaptic NT secretion for synaptic plasticity. Here, we transfected cultures of dissociated rat hippocampal neurons with green fluorescent protein-tagged versions of BDNF and NT-3, respectively, and identified NT vesicles at glutamatergic synapses by colocalization with the cotransfected postsynaptic marker PSD-95 (postsynaptic density-95)-DsRed. Depolarization-induced secretion of BDNF and NT-3 was monitored with live cell imaging. Direct postsynaptic depolarization with elevated K+ in the presence of blockers of synaptic transmission allowed us to investigate the signaling cascades that are involved in the postsynaptic NT vesicle secretion process. We show that depolarization-induced postsynaptic NT secretion is elicited by Ca2+ influx, either via L-type voltage-gated calcium channels or via NMDA receptors. Subsequent release of Ca2+ from internal stores via ryanodine receptors is required for the secretion process. Postsynaptic NT secretion is inhibited in the presence of KN-62 ([4(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester) and KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide), indicating a critical dependence on the activation of alpha-calcium-calmodulin-dependent protein kinase II (CaMKII). The cAMP/protein kinase A (PKA) signaling inhibitor Rp-cAMP-S impaired NT secretion, whereas elevation of intracellular cAMP levels was without effect. Using the Trk inhibitor k252a, we show that NT-induced NT secretion does not contribute to the NT release process at synapses, and BDNF does not induce its own secretion at postsynaptic sites. Release experiments in the presence of the fluorescence quencher bromphenol blue provide evidence for asynchronous and prolonged fusion pore opening of NT vesicles during secretion. Because fusion pore opening is fast compared with compound release, the speed of NT release seems to be limited by diffusion of NTs out of the vesicle. Together, our results reveal a strong dependence of activity-dependent postsynaptic NT secretion on Ca2+ influx, Ca2+ release from internal stores, activation of CaMKII, and intact PKA signaling, whereas Trk signaling and activation of Na+ channels is not required.

NT-3 modulates BDNF and proBDNF levels in naïve and kindled rat hippocampus.

Both mature and precursor forms of neurotrophins regulate nerve development, survival and plasticity. Brain-derived neurotrophic factor (BDNF) synthesis and secretion in turn are regulated by neuronal activity, such as epilepsy. Further, neurotrophins themselves are regulated by neurotrophin levels. Neurotrophin-3 (NT-3) and BDNF in particular can be co-expressed and each can regulate the levels of the other. This regulation is thought to be mediated through receptor tyrosine kinase (Trk) activity. It is not known whether this neurotrophin-neurotrophin interaction occurs in hippocampal tissue in vivo, or how it is influenced by neuronal activation. In this study, we explored the reciprocal influences of intraventricular infusions of NT-3 and BDNF in naïve and kindled hippocampi of rats using Western blotting. We confirm that hippocampal kindling resulted in a significant increase in levels of BDNF both in cytochrome C (control) infused and NT-3 infused kindled rats. However, NT-3 infusion significantly reduced BDNF levels in both kindled and non-kindled hippocampi compared to their cytochrome C infused counterparts. These results are consistent with our earlier studies demonstrating lowered levels of TrkA and TrkC (NGF modulates BDNF levels via TrkA) following chronic NT-3 infusion. Although kindling led to an increase in BDNF, this was not accompanied by any detectable change in the levels of proBDNF. However, there was a significant increase in proBDNF following NT-3 infusions, suggesting NT-3 may reduce proBDNF processing. In contrast, neither NT-3 nor proNT-3 levels were affected by kindling or chronic BDNF infusions, consistent with down-regulation of TrkB by chronic BDNF infusion. Thus, modulation of BDNF by NT-3, likely mediated by Trk receptors, occurs in naïve and kindled adult rat hippocampus.

Embryonic sympathoblasts transiently express TrkB in vivo and proliferate in response to brain-derived neurotrophic factor in vitro.

BACKGROUND: Nerve growth factor and neurotrophin-3 are involved in the development of sympathetic neurons; however, whether brain derived neurotrophic factor also plays a role is not known. The purpose of this study was to determine whether BDNF and its receptor, TrkB, are expressed during the development of paravertebral sympathetic ganglia in vivo and to determine the effect of BDNF in vitro. RESULTS: As neural crest cells coalesce to form sympathetic ganglia, TrkB-positive cells are seen in both chicken and mouse embryos. In chicken embryos, TrkB-expressing cells first appear at Hamburger-Hamilton Stage (St) 27 and they co-express HNK-1, confirming that they are migrating neural crest cells. The TrkB-positive cells lack neural markers at this stage; however, they migrate with other neurally differentiating cells that are TrkA and TrkC-positive. By St. 29/30, TrkB-positive cells begin to express the neural specific markers Hu C/D and Islet-1; eventually, all TrkB positive cells commence neural differentiation. By St. 34, TrkB and TrkC staining are lost. BDNF transcript expression parallels that of TrkB. In the mouse, TrkB-positive cells surround newly formed sympathetic ganglia and a small number of TrkB positive cells that co-express tyrosine hydroxylase are seen within ganglia between E13.5-15. In cell culture, many cells from St. 29-30 chicken lumbar sympathetic ganglia express neural markers and are dividing, indicating that they are sympathoblasts. Sympathoblasts and neurons require both nerve growth factor and neurotrophin-3 for survival. BDNF increases the number of cells expressing neural markers in culture by increasing number of cells that incorporate bromodeoxyuridine. In contrast, most TrkB-positive sympathetic cells in vivo are not actively proliferating between E6-E8. CONCLUSION: Developing paravertebral sympathetic ganglia in avian and murine embryos contain a subpopulation of sympathoblasts that transiently express TrkB and ultimately commence neuronal differentiation. These TrkB expressing sympathoblasts are not actively dividing in vivo; yet, when placed in vitro, will divide in response to BDNF. This suggests that the availability of BDNF in vivo fails to reach a threshold necessary to induce proliferation. We suggest that excess TrkB stimulation of sympathoblasts in vivo may lead to the genesis of neuroblastoma.

Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury.

STUDY DESIGN: An animal model of transected spinal cord injury (SCI) was used to test the hypothesis that cografted neural stem cells (NSCs) and NT-3-SCs promote morphologic and functional recoveries of injured spinal cord. OBJECTIVE: To explore whether cotransplant of NSCs and NT-3-SCs could promote the injured spinal cord repair. SETTING: Zhongshan Medical College, Sun Yat-sen University, PR China. METHODS: Female Sprague-Dawley (SD) rats weighing on 200-220 g were used to prepare SCI models. The spinal cord was transected between T(9) and T(10), then NSCs, SCs+NSCs, LacZ-SCs+NSCs, or NT-3-SCs+NSCs were grafted into the transected site. RESULTS: (1) Part of NSCs could differentiate to neuron-like cells in the transected site and the percentage of differentiation was NT-3-SCs+NSCs group>SCs+NSCs group>NSCs group. (2) In the grafted groups, there were 5-HT, CGRP, and SP positive nerve fibres within the transected site. Some fluorogold (FG)-labeled cells were found in the spinal cord rostral to the transected site, the red nuclei and the inner pyramidal layer of sensorimotor cortex. (3) The cells grafted could enhance the injured neurons survival in inner pyramidal layer of sensorimotor cortex, red nuclei of midbrain, and Clark's nuclei of spinal cord's L1 segment, could decrease the latency and increase the amplitude of cortical somatosensory evoked potential (CSEP) and cortical motor evoked potential (CMEP), and could promote partly structural and functional recovery of the SCI rats. CONCLUSION: These results demonstrate that cografted NT-3-SCs and NSCs is a potential therapy for SCI. SPONSORSHIP: This research was supported by Chinese National Key Project for Basic Research (G1999054009), Chinese National Natural Science Foundation (30270700) and Social Developmental Foundation of Guangdong Province (2003C33808) to YS Zeng; Natural Science Foundation of Guangdong Province (04300468) and Medical Science Research Grant of Guangdong Province (A2004081) to JS Guo.