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A mild ischemic stroke may cause both debilitating locomotor and cognitive decline, for which the mechanism is not fully understood, and no therapies are currently available. In this study, a nonfatal stroke model was constructed in mice by a modified middle cerebral artery occlusion (MCAO) procedure, allowing an extended recovery period up to 28 days. The extended MCAO model successfully mimicked phenotypes of a recovery phase post-stroke, including locomotor motor and cognitive deficiencies, which were effectively improved after Shuxuening injection (SXNI) treatment. Tissue slices staining showed that SXNI repaired brain injury and reduced neuronal apoptosis, especially in the hippocampus CA3 region. Transcriptomics sequencing study revealed 565 differentially expressed genes (DEGs) in the ischemic brain after SXNI treatment. Integrated network pharmacological analysis identified Neurotrophin/Trk Signaling was the most relevant pathway, which involves 15 key genes. Related DEGs were further validated by RT-PCR. Western-blot analysis showed that SXNI reversed the abnormal expression of BDNF, TrkB, Mek3 and Jnk1after stroke. ELISA found that SXNI increased brain level of p-Erk and Creb. At sub-brain level, the expression of BDNF and TrkB was decreased and GFAP was increased on the hippocampal CA3 region in the post-stroke recovery phase and this abnormality was improved by SXNI. In vitro experiments also found that oxygen glucose deprivation reduced the expression of BDNF and TrkB, which was reversed by SXNI. In summary, we conclude that SXNI facilitates the recovery of cognitive and locomotor dysfunction by modulating Neurotrophin/Trk Signaling in a mouse model for the recovery phase of post-ischemic stroke., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Dehydroepiandrosterone (DHEA) is the most abundant circulating steroid hormone in humans, produced by the adrenals, the gonads and the brain. DHEA was previously shown to bind to the nerve growth factor receptor, tropomyosin-related kinase A (TrkA), and to thereby exert neuroprotective effects. Here we show that DHEA reduces microglia-mediated inflammation in an acute lipopolysaccharide-induced neuro-inflammation model in mice and in cultured microglia in vitro. DHEA regulates microglial inflammatory responses through phosphorylation of TrkA and subsequent activation of a pathway involving Akt1/Akt2 and cAMP response element-binding protein. The latter induces the expression of the histone 3 lysine 27 (H3K27) demethylase Jumonji d3 (Jmjd3), which thereby controls the expression of inflammation-related genes and microglial polarization. Together, our data indicate that DHEA-activated TrkA signaling is a potent regulator of microglia-mediated inflammation in a Jmjd3-dependent manner, thereby providing the platform for potential future therapeutic interventions in neuro-inflammatory pathologies.

The binding of the human nerve growth factor (NGF) protein to tropomyosin receptor kinase A (TrkA) is associated with Alzhemeir's development. Owing to the large presence of zinc(II) ions in the synaptic compartments, the zinc ions might be bound to the complex in vivo. Here, we have identified a putative zinc binding site using a combination of computations and experiments. First, we have predicted structural features of the NGF/TrkA complex in an aqueous solution by molecular simulation. Metadynamics free energy calculations suggest that these are very similar to those in the X-ray structure. Here, the "crab" structure of the NGF shape binds tightly to two TrkA "pincers". Transient conformations of the complex include both more extended and more closed conformations. Interestingly, the latter features facial histidines (His60 and His61) among the N-terminal D1-D3 domains, each of which is a potential binding region for biometals. This suggests the presence of a four-His Zn binding site connecting the two chains. To address this issue, we investigated the binding of a D1-D3 domains' peptide mimic by stability constant and nuclear magnetic resonance measurements, complemented by density functional theory-based calculations. Taken together, these establish unambiguously a four-His coordination of the metal ion in the model systems, supporting the presence of our postulated binding site in the NGF/TrkA complex.

Background: Pyrrolizidine alkaloids (PAs) are commonly found in many plants including those used in medical therapeutics. The hepatotoxicities of PAs have been demonstrated both in vivo and in vitro; however, the neurotoxicities of PAs are rarely mentioned., Purpose: In this study, we aimed to investigate in vitro neurotoxicities of clivorine, one of the PAs found in various Ligularia species, in cultured PC12 cells., Study Design: PC12 cell line was employed to first elucidate the neurotoxicity and the underlying mechanism of clivorine, including cell viability and morphology change, neuronal differentiation marker and signaling pathway., Methods: PC12 cells were challenged with series concentrations of clivorine and/or nerve growth factor (NGF). The cell lysates were collected for MTT assay, trypan blue staining, immunocytofluorescent staining, qRT-PCR and western blotting., Results: Clivorine inhibited cell proliferation and neuronal differentiation evidenced by MTT assay and dose-dependently reducing neurite outgrowth, respectively. In addition, clivorine decreased the level of mRNAs encoding for neuronal differentiation markers, e.g. neurofilaments and TrkA (NGF receptor). Furthermore, clivorine reduced the NGF-induced the phosphorylations of TrkA, protein kinase B and cAMP response element-binding protein in cultured PC12 cells., Conclusion: Taken together, our results suggest that clivorine might possess neurotoxicities in PC12 cells via down-regulating the NGF/TrkA/Akt signaling pathway. PAs not only damage the liver, but also possess neurotoxicities, which could possibly result in brain disorders, such as depression., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Tricyclic antidepressant amitriptyline (AM) has been shown to exert neurotrophic activity on neurons. We thus explored whether AM may aid the neuronal development and protect anesthesia-induced neuro-injury in young spinal cord dorsal root ganglion (DRG) neurons.The DRG explants were prepared from 1-day-old rats. The effect of AM on aiding DRG neural development was examined by immunohistochemistry at dose-dependent manner. AM-induced changes in gene and protein expressions, and also phosphorylation states of tyrosine kinases receptor A (TrkA) and B (TrkB) in DRG, were examined by quantitative real-time polymerase chain reaction and western blot. The effect of AM on attenuating lidocaine-induced DRG neurodegeneration was examined by immunohistochemistry, and small interfering RNA (siRNA)-mediated TrkA/B down-regulation.Amitriptyline stimulated DRG neuronal development in dose-dependent manner, but exerted toxic effect at concentrations higher than 10 M. AM activated TrkA in DRG through phosphorylation, whereas it had little effect on TrkB-signaling pathway. AM reduced lidocaine-induced DRG neurodegeneration by regenerating neurites and growth cones. Moreover, the neuroprotection of AM on lidocaine-injured neurodegeneration was blocked by siRNA-mediated TrkA down-regulation, but not by TrkB down-regulation.Amitriptyline facilitated neuronal development and had protective effect on lidocaine-induced neurodegeneration, very likely through the activation of TrkA-signaling pathway in DRG.

Clioquinol is considered to be a causative agent of subacute myelo-optico neuropathy (SMON), although the pathogenesis of SMON is yet to be elucidated. We have previously shown that clioquinol inhibits nerve growth factor (NGF)-induced Trk autophosphorylation in PC12 cells transformed with human Trk cDNA. To explore the further mechanism of neuronal damage by clioquinol, we evaluated the acetylation status of histones in PC12 cells. Clioquinol reduced the level of histone acetylation, and the histone deacetylase (HDAC) inhibitor Trichostatin A upregulated acetylated histones and prevented the neuronal cell damage caused by clioquinol. In addition, treatment with HDAC inhibitor decreased neurite retraction and restored the inhibition of NGF-induced Trk autophosphorylation by clioquinol. Thus, clioquinol induced neuronal cell death via deacetylation of histones, and HDAC inhibitor alleviates the neurotoxicity of clioquinol. Clioquinol is now used as a potential medicine for malignancies and neurodegenerative diseases. Therefore, HDAC inhibitors can be used as a candidate medicine for the prevention of its side effects on neuronal cells., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

In this study, we evaluated, in the mouse, the effects of 20 mg/kg i.p. daily administration for 15 consecutive days of a blend of polyphenols, containing mostly oleuropein, extracted from the olive leaves (Olea europaea) on brain nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and on the expression of their receptors, TrkA, TrkB and p75. Polyphenols decreased the levels of reduced glutathione (GSH) and increased the levels of NGF and BDNF in the serum. In the brain, we found decreased levels of NGF and BDNF in the hippocampus and striatum but elevated levels of NGF in the olfactory lobes and hypothalamus and again BDNF potentiation in the olfactory lobes. No changes in TrkA, TrkB and p75 expression were observed. In conclusion, olive polyphenols may not only elicit an activation of the rodent olfactory system by increasing the levels of NGF and BDNF but also be stressing for the animal by reducing both the levels of hippocampal NGF/BDNF and serum GSH and increasing serum levels of NGF and BDNF.

p75(NTR) is a neurotrophin receptor that can mediate either survival or death of neurons depending on the cell context. Modulation of p75(NTR) is a promising strategy to promote neuronal survival for treatment of cognitive disorders such as Alzheimer's disease. Despite years of investigation into the signaling mechanisms of p75(NTR), no p75(NTR) signaling assay has yet been developed that is compatible with efficient screening of small-molecule modulators. In this work, we developed a homogeneous cell-based assay for screening p75(NTR) modulators and studying p75(NTR) function. Stimulation of p75(NTR)-transfected cells using either nerve growth factor (NGF) or Pro-NGF resulted in an enhanced caspase-3 activity as assessed by cleavage of a fluorescent caspase-3 substrate. Optimization of the assay with respect to time, cell density, NGF and Pro-NGF concentration, and other factors provided a twofold increase in the caspase-3 activity compared to background. Withdrawal of serum during the NGF or Pro-NGF treatment period was found to be essential for p75(NTR)-dependent caspase-3 activation. We validated the method by demonstrating that a signaling-incompetent p75(NTR) mutant could not substitute for wild-type p75(NTR) in mediating caspase-3 activation. A focused library screen identified new inhibitors of p75(NTR) signaling. This method will be useful for identifying small-molecule modulators of p75(NTR) as well as further characterizing downstream signaling events.

Aim: To investigate the effect of Ad-ING4 on proliferation and migration of glioma cells and explore its probable mechanism., Methods: U251 were infected with Ad-ING4. ING4 gene expression was evaluated by RT-PCR. MTT assay was adopted to evaluate the effect of ING4 on proliferation of U251; Boyden chamber assay was used to check the effect of ING4 on the migration of U251. In ING4 transfected U251, Western blot was used for detecting NGF and TrkA expression; Pull-down assay was used for detecting active RhoA expression., Results: ING4 was overexpressed in Ad-ING4 transfected U251 cells. ING4 inhibited proliferation and migration of U251 significantly. Moreover, overexpression of ING4 result in depression of NGF, TrkA and active RhoA., Conclusion: ING4 mediated inhibition of the proliferation and migration of human glioma cells by down regulating NGF, TrkA and active RhoA expression.

Apolipoprotein E (apoE) is one of the major transporters of cholesterol in the body and is essential for maintaining various neural functions in the brain. Given that hypercholesterolemia is a risk factor in Alzheimer's disease (AD), it has been suggested that altered cholesterol metabolism may be involved in the development of the pathogenesis, including neural degeneration, commonly observed in AD patients. Neurotrophic factors and their receptors, which are known to regulate various neural functions, are also known to be altered in various neurodegenerative diseases. We therefore hypothesized that cholesterol metabolism may itself influence the neurotrophin system within the brain. We decided to investigate this possibility by modulating the amount of dietary cholesterol given to apoE-knockout (apoE-KO) and wild-type (WT) mice, and examining the mRNA expression of various neurotrophin ligands and receptors in their hippocampal formations. Groups of eight-week-old apoE-KO and WT mice were fed a diet containing either "high" (HCD) or "normal" (ND) levels of cholesterol for a period of 12 weeks. We found that high dietary cholesterol intake elevated BDNF mRNA expression in both apoE-KO and WT mice and TrkB mRNA expression in apoE-KO animals. On the other hand, NGF and TrkA mRNA levels remained unchanged irrespective of both diet and mouse type. These findings indicate that altered cholesterol metabolism induced by HCD ingestion combined with apoE deficiency can elicit a differential response in the various neurotrophin ligand/receptor systems in the mouse hippocampus. Whether such changes can lead to neural degeneration, and the mechanisms that may be involved in this, awaits further research.

Brain-derived neurotrophic factor (BDNF) is closely linked with neuronal survival and plasticity in psychiatric disorders. In this work, we engineered degradable, injectable alginate microspheres and non-degradable, implantable poly(ethylene vinyl acetate) matrices to continuously deliver BDNF to the dorsal hippocampus of rats for two days or more than a week, respectively. The antidepressant-like behavioral effects of BDNF delivery were examined in the Porsolt forced swim test. Rats were sacrificed 10days after surgery and tissue samples were analyzed by western blot. A small dose of BDNF delivered in a single infusion, or from a two-day sustained-release alginate implant, produced an antidepressant-like behavior, whereas the same dose delivered over a longer period of time to a larger tissue region did not produce antidepressant-like effects. Prolonged delivery of BDNF resulted in a dysregulation of plasticity-related functions: increased dose and duration of BDNF delivery produced increased levels of TrkB, ERK, CREB, and phosphorylated ERK, while also producing decreased phosphorylated CREB. It is evident from this work that both duration and magnitude of BDNF dosing are of critical importance in achieving functional outcome., (2010 Elsevier B.V. All rights reserved.)

A conditioning lesion improves regeneration of central and peripheral axons of dorsal root ganglion (DRG) neurons after a subsequent injury by enhancing intrinsic growth capacity. This enhanced growth state is also observed in cultured DRG neurons, which support a more sparsely and rapidly elongating mode of growth after a prior conditioning lesion in vivo. Here we examined differences in the capacity or requirements of specific types of sensory neurons for regenerative growth, which has important consequences for development of strategies to improve recovery after injury. We showed that after partial or complete injury of the sciatic nerve in mice, an elongating mode of growth in vitro was activated only in DRG neurons that did not express calcitonin gene-related peptide (CGRP) or bind Bandeiraea simplicifolia I-isolectin B4 (IB4). We also directly examined the response of conditioned sensory neurons to nerve growth factor (NGF), which does not enhance growth in injured peripheral nerves in vivo. We showed that after partial injury, NGF stimulated a highly branched and linearly restricted rather than elongating mode of growth. After complete injury, the function of NGF was impaired, which immunohistochemical studies of DRG indicated was at least partly due to downregulation of the NGF receptor, tropomyosin-related kinase A (TrkA). These results suggest that, regardless of the type of conditioning lesion, each type of DRG neuron has a distinct intrinsic capacity or requirement for the activation of rapidly elongating growth, which does not appear to be influenced by NGF., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75(NTR)), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75(NTR) and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75(NTR) and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75(NTR)/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75(NTR) or TrkA. Interestingly, immunoreactivity to anti-p75(NTR) antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75(NTR), when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75(NTR) is turned on.

Background: Nerve growth factor (NGF) is an important mediator of airway hyper-responsiveness and hyperalgesia but its role in cough is unknown., Objectives: In this study the effects of NGF on the cough reflex and airway calibre were investigated in guinea pigs. The involvement of the tropomyosin-related kinase A (TrkA) receptor and transient receptor potential vanilloid-1 (TRPV1), and the p38 mitogen-activated protein kinase (MAPK)-dependent pathway in any NGF-induced effects on cough and airway obstruction was also assessed., Methods: Guinea pigs were placed in a transparent whole-body plethysmograph box. Cough was assessed visually, acoustically and by analysis of the airflow signal. Airway obstruction was measured using enhanced pause (Penh) as an index., Results: Exposure of guinea pigs to NGF did not induce a cough response nor a significant airway obstruction. However, exposure of guinea pigs to NGF immediately before citric acid inhalation resulted in a significant increase in the citric acid-induced cough and airway obstruction compared with vehicle-treated animals. Pretreatment with the TrkA receptor antagonist, K252a, or the TRPV1 antagonist, iodoresiniferatoxin, significantly inhibited the NGF-enhanced cough and airway obstruction. Exposure to NGF also increased p38 MAPK phosphorylation, but pretreatment with the p38 MAPK inhibitor, SB203580, did not affect either the NGF-enhanced cough or airway obstruction despite preventing the NGF-induced elevation in p38 MAPK phosphorylation., Conclusions: The data show that NGF can enhance both cough and airway obstruction via a mechanism that involves the activation of the TrkA receptor and TRPV1 but not the p38 MAPK-dependent pathway.

In this study, possible involvements of choline and nicotinic acetylcholine receptors (nAChRs) in neurotrophic-related neuronal plasticity were investigated. Primary cell cultures from rat cerebral cortex were exposed for 72 h to the alpha7 nAChR selective agonist choline and protein expression levels of the neurotrophin receptors p75, TrkA, TrkB and TrkC were examined. The results revealed a choline-induced attenuation of the TrkB expression, whereas the other neurotrophin receptors were not affected. Further analysis of choline-exposed cell cultures showed an increased protein level of the TrkB ligand brain-derived neurotrophic factor (BDNF). This increase was obtained in cell cultures where the alpha7 nAChR subunit was detected, but not in younger cell cultures where this subunit could not be detected. It is speculated that a choline-induced change of alpha7 nAChRs activity may have resulted in the observed increase of BDNF level and down-regulation of the TrkB receptor.

Background: To date, no effective therapeutic treatment prevents prostate cancer (PCa) progression to more advanced and invasive disease forms. It has been demonstrated that the simultaneous high expression of p185(HER2) and TrkA might confer a proliferative advantage to PCa cells., Methods: In this work we verified the crosstalk between TrkA and Her2 signaling pathways and the effects of a combined treatment with Her2 and TrkA inhibitors., Results: NGF induced TrkA activation and stimulated cell proliferation of PCa cells. NGF induced also tyrosine phosphorylation of p185(HER2). This event was only partially inhibited by the pan Trk inhibitor, CEP-701 but was strongly blocked by pertuzumab, a humanized antibody blocking Her2 heterodimerization. In presence of NGF, TrkA and Her2 co-precipitated and this was dependent to the relative high cellular levels of TrkA since when cell lysates were immunoprecipitated with an antibody against Her2 the amount of TrkA were proportional to the cellular levels of this receptor. On the contrary when we immunoprecipitated using an antibody against TrkA the amount of Her2 seemed independent to cellular levels of Her2. So, combined treatment between CEP-701 and pertuzumab showed supra-additive effects in cells with higher levels of TrkA and Her2 suggesting once again that this was indicative of a higher response to treatment., Conclusions: Our data suggest that the dual inhibition of TrkA and Her2 may be useful in a subset of patients in which TrkA and Her2 are overexpressed and in which the possibility of TrkA and Her2 protein-binding is elevated., (2008 Wiley-Liss, Inc.)

Laminin-1, an extracellular matrix molecule, promotes neurite outgrowth through the interaction of integrin and actin. Monosialoganglioside GM1 in the lipid rafts associates with and activates the NGF receptor TrkA, and enhances neurite outgrowth. However, the role of GM1 in laminin-1-induced neurite outgrowth was still unclear. Here, we describe that laminin-1 binds to GM1 through a carbohydrate moiety and a specific conformation of GM1, induces focal formation of large clusters of GM1, and enhances the relocation of TrkA in the membrane of dorsal root ganglion (DRG) and PC12 cells. We found that laminin-1-mediated clustering of GM1 causes the translocation and enrichment of beta1 integrin in lipid rafts--where TrkA colocalizes with beta1 integrin--and the activation of Lyn, Akt and MAPK to promote the outgrowth of neurites. Our results suggest that the binding of laminin-1 to GM1 facilitates the formation of a focal microdomain in the membrane, and enhances signal transduction that promotes neurite outgrowth by linking NGF-TrkA signaling with the laminin-integrin signaling pathways.

Misdirection of axons after nerve injury impairs successful regeneration of adult neurons. Investigations of axon guidance in development have provided an understanding of pathfinding, but their relevance to regenerating adult axons is unclear. We investigated adult mammalian axon guidance during regeneration after peripheral nerve injury and focused on the effects of the prototypic guidance molecule nerve growth factor (NGF). Adult rat sensory neurons from dorsal root ganglia that expressed the NGF receptor tropomyosin-related kinase A (trkA) were presented with a point source of NGF in vitro. Naive trkA neurons had no net turning response to NGF, but if they had been preconditioned by a peripheral nerve transection in vivo before culturing, their growth cones were attracted toward the NGF gradient. A laminin substrate was required for this behavior and an anti-trkA antibody interrupted turning. These data demonstrate that injured adult mammalian axons can be guided as they regenerate. Moreover, despite the downregulation of trkA mRNA and protein levels within the dorsal root ganglion after injury, sensory neurons retain and increase trkA protein at the injury site where the regenerating axons are found. This may enhance the axonal response to NGF and allow guidance along an NGF gradient created in vivo in the distal nerve stump.

Lead is a potent developmental neurotoxicant that affects many aspects of cognition and behavior. The hippocampus and striatum are among the areas particularly sensitive to the effects of lead and cholinergic neurons in both regions depend upon nerve growth factor (NGF) for their survival and maturation. The present study examined the extent to which postnatal lead exposure may affect the survival and expression of neuroptrophin receptors of septo-hippocampal cholinergic projection neurons in the medial septum/vertical limb of the diagonal band of Broca (MS/VDB) and cholinergic neurons of the striatum. Weanling rats were fed chow containing lead acetate for 30 days and effects on cholinergic cell number and the number of cells expressing neurotrophin receptors p75(NGFR) and trkA were assessed. A decrease in the number of cells expressing p75(NGFR) and an increase in the number of cells expressing trkA receptor was observed in the MS/VDB of lead-exposed rats, without a loss of cholinergic cell number or alteration in cell size. Lead-exposure resulted in a significant decrease in trkA-expressing cells in the striatum but no change in the number or size of cholinergic neurons. These results suggest that a brief postnatal lead exposure does not result in loss of MS/VDB or striatal cholinergic neurons but does modify the expression of neurotrophin receptors in these regions. The significance of these effects on the septo-hippocampal and striatal functioning remains to be studied.

The primary therapeutic agents used for schizophrenia, antipsychotic drugs, ameliorate psychotic symptoms; however, their chronic effects on cognition (or the physiologic processes of the brain that support cognition) are largely unknown. The purpose of this rodent study was to extend our previous work on this subject by investigating persistent effects (i.e. during a 14 day drug-free washout period) of chronic treatment (i.e. ranging from 45 days to 6 months) with a representative first and second generation antipsychotic. Drug effects on learning and memory and important neurobiological substrates of memory, the neurotrophin, nerve growth factor (NGF) and its receptors, and certain components of the basal forebrain cholinergic system were investigated. Behavioral effects of oral haloperidol (2.0 mg/kg/day), or risperidone (2.5 mg/kg/day) were assessed in an open field, a water maze task, and a radial arm maze procedure and neurochemical effects in brain tissue were subsequently measured by enzyme-linked immunosorbent assays (ELISAs). The results indicated that both antipsychotics produced time-dependent and protracted deficits in the performance of a water maze procedure when compared with vehicle-treated controls, while neither drug was associated with significant alterations in radial arm maze performance. Interestingly, haloperidol, but not risperidone, was detectible in the rodent brain in appreciable levels for up to 2 weeks after drug discontinuation. Both antipsychotics were also associated with reduced levels of NGF protein in the basal forebrain and prefrontal cortex and significant (or nearly significant) decreases in phosphorylated tropomyosin-receptor kinase A (TrkA) protein and the vesicular acetylcholine transporter (depending on the brain region analyzed). Neither antipsychotic markedly affected TrkA or p75 neurotrophin receptor levels. These data in rats indicate that chronic treatment with either haloperidol or risperidone may be associated with protracted negative effects on cognitive function as well as important neurotrophin and neurotransmitter pathways that support cognition.

Activation and mobilization of microglia are early events in the majority of brain pathologies. Among the signalling molecules that can affect microglial behaviour, we investigated whether nerve growth factor (NGF) was able to influence microglial motility. We found that NGF induced chemotaxis of microglial cells through the activation of TrkA receptor. In addition, NGF chemotactic activity was increased in the presence of low concentrations (< or =0.2 ng/ml) of transforming growth factor-beta (TGF-beta), which at this concentration showed chemotactic activity per se. On the contrary, NGF-induced microglial migration was reduced in the presence of chemokinetic concentration of TGF-beta (> or =2 ng/ml). Finally, both basal and NGF-induced migratory activity of microglial cells was increased after a long-term exposure of primary mixed glial cultures to 2 ng/ml of TGF-beta. Our observations suggest that both NGF and TGF-beta contribute to microglial recruitment. The chemotactic activities of these two pleiotropic factors could be particularly relevant during chronic diseases in which recruited microglia remove apoptotic neurons in the absence of a typical inflammatory reaction.

The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death.

The current study determined the ability of a p75(NTR) antagonistic cyclic peptide to rescue cells from beta amyloid (Abeta) (1-40)-induced death. p75(NTR)-, p140(trkA)-NIH-3T3 cells or E17 foetal rat cortical neurones were incubated with 125I-NGF or 125I-Abeta (1-40) and increasing concentrations of the cyclic peptide (CATDIKGAEC). Peptide ability to displace 125I-NGF or 125I-Abeta (1-40) binding was determined. Duplicate cultures were preincubated with CATDIKGAEC (250 nM) or diluent and then stimulated with Abeta (1-40). Peptide ability to displace Abeta (1-40) binding, interfere with Abeta (1-40)-induced signalling and rescue cells from Abeta-mediated toxicity was determined by immunoprecipitation and autoradiography, Northern blotting, JNK activation, MTT and trypan blue assays. The peptide inhibited NGF and Abeta (1-40) binding to p75(NTR), but not to p140(trkA). Abeta (1-40) induced c-jun transcription (57.3% +/- 0.07%) in diluent-treated p75(NTR)-cells, but not in cells preincubated with the cyclic peptide. Also, at 250 nM, the peptide reduced Abeta (1-40)-induced phosphorylation of JNK by 71.8% +/- 0.03% and protected neurones against Abeta-induced toxicity as determined by: trypan blue exclusion assay (53% +/- 11% trypan blue-positive cells in diluent pretreated cultures vs. 28% +/- 5% in cyclic peptide-pretreated cultures); MTT assay (0.09 +/-0.03 units in diluent-pretreated cells vs. 0.12 +/- 0.004 units in cyclic peptide-pretreated cells); and visualization of representative microscopic fields. Our data suggest that a cyclic peptide homologous to amino acids 28-36 of NGF known to mediate binding to p75(NTR) can interfere with Abeta (1-40) signalling and rescue neurones from Abeta (1-40)-induced toxicity.

There is a great deal of interest in neurotrophin therapy to prevent neuronal degeneration. However, the blood-brain barrier presents a major hurdle in the use of peptide therapeutics. The goal of this study was to identify small molecule, cell-permeable nerve growth factor (NGF) activators. Combinatorial libraries of asterriquinones (>300) and mono-indolyl-quinones (>60) were screened using a 96-well enzyme-linked immunosorbent assay that detects phosphorylated TrkA, the NGF receptor. The libraries were also screened for dose-dependent cytotoxicity. From these screens, we generated quantitative structure-activity relationship models for activity and toxicity, and then we selected two compounds, 2-(6-chloro-1H-indol-3-yl)-5-(2-cyclopropyl-1H-indol-3-yl)-3,6-dihydroxy-[1,4]benzoquinone (1H5) and 2,5-dimethoxy-3-(7-fluoro-1H-indol-3-yl)-[1,4]-benzoquinone (5E5), for further study based on high activity and low toxicity. Compound 1H5 (30 microM) is an asterriquinone that is a moderate TrkA activator (50% the activity of 100 ng/ml NGF), and it shows little toxicity at concentrations up to 100 microM. 1H5 can protect differentiated PC12 neurons from apoptotic cell death induced by NGF withdrawal. Compound 5E5 (30 microM) is a mono-indolyl-quinone that is a very strong activator of TrkA (>200% the activity of 100 ng/ml NGF), and it is nontoxic at concentrations up to 10 microM. Activation of TrkA can be detected at 1 microM 5E5, and 3 to 10 microM 5E5 activates TrkA and extracellular signal-regulated kinase as strongly as a maximal dose of NGF (100 ng/ml). A combination of a low dose of 5E5 (1 microM) with a submaximal dose of NGF (10 ng/ml) promotes neuronal differentiation of PC12 cells. These compounds represent a new class of TrkA activators that could have potential utility in the treatment of neurodegenerative diseases.

Neuroblastoma (NB) often causes spontaneously regression, and can mature to ganglioneuroma. The form with the most favorable prognosis expresses high levels of TrkA, a high-affinity receptor for nerve growth factor (NGF), whereas advanced NB and associated cell lines have abnormalities in the NGF/TrkA signaling pathway. A novel cyclophane, cyclophane pyridine (CPPy), was designed to conserve the tyrosine phosphorylation of TrkA, thereby enhancing NGF/TrkA signal transduction. We investigated whether this compound improved NGF-induced tyrosine phosphorylation of the Y490 domain of TrkA and conserved the expression of an early gene (c-fos) in human NB cell lines (IMR-32 and NB-39). As determined by Western blotting, TrkA (Y490) phosphorylation was enhanced by the combination of CPPy (10(-8) M) and NGF (100 ng/ml) compared with NGF alone. CPPy also conserved NGF-induced c-fos mRNA expression. Moreover, CPPy induced the morphological differentiation of NB cells, leading to expression of the neuronal marker gene GAP-43. These data suggest that CPPy can induce the differentiation of NB cell lines by facilitating NGF-induced TrkA/Ras/MAPK signal transduction, and may therefore be an effective therapeutic agent for NB.

GIPC is a PDZ protein located on peripheral endosomes that binds to the juxtamembrane region of the TrkA nerve growth factor (NGF) receptor and has been implicated in NGF signaling. We establish here that endogenous GIPC binds to the C terminus of APPL, a Rab5 binding protein, which is a marker for signaling endosomes. When PC12(615) cells are treated with either NGF or antibody agonists to activate TrkA, GIPC and APPL translocate from the cytoplasm and bind to incoming, endocytic vesicles carrying TrkA concentrated at the tips of the cell processes. GIPC, but not APPL, dissociates from these peripheral endosomes prior to or during their trafficking from the cell periphery to the juxtanuclear region, where they acquire EEA1. GIPC's interaction with APPL is essential for recruitment of GIPC to peripheral endosomes and for TrkA signaling, because a GIPC PDZ domain mutant that cannot bind APPL or APPL knockdown with small interfering RNA inhibits NGF-induced GIPC recruitment, mitogen-activated protein kinase activation, and neurite outgrowth. GIPC is also required for efficient endocytosis and trafficking of TrkA because depletion of GIPC slows down endocytosis and trafficking of TrkA and APPL to the early EEA1 endosomes in the juxtanuclear region. We conclude that GIPC, following its recruitment to TrkA by APPL, plays a key role in TrkA trafficking and signaling from endosomes.

The present study was undertaken to evaluate the implication of delta-opioid receptor function in neurogenesis and neuroprotection. We found that the stimulation of delta-opioid receptors by the selective delta-opioid receptor agonist SNC80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide] (10 nm) promoted neural differentiation from multipotent neural stem cells obtained from embryonic C3H mouse forebrains. In contrast, either a selective micro-opioid receptor agonist, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), or a specific kappa-opioid receptor agonist, (-)-trans-(1S,2S)-U-50488 hydrochloride (U50,488H), had no such effect. In addition to neural differentiation, the increase in cleaved caspase 3-like immunoreactivity induced by H2O2 (3 microm) was suppressed by treatment with SNC80 in cortical neuron/glia co-cultures. These effects of SNC80 were abolished by a Trk-dependent tyrosine kinase inhibitor: (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(cde)trinden-1-one (K-252a). The SNC80-induced neural differentiation was also inhibited by treatment with the protein kinase C (PKC) inhibitor, phosphatidylinositol 3-kinase (PI3K) inhibitor, mitogen-activated protein kinase kinase (MEK) inhibitor or Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These findings raise the possibility that delta-opioid receptors play a crucial role in neurogenesis and neuroprotection, mainly through the activation of Trk-dependent tyrosine kinase, which could be linked to PI3K, PKC, CaMKII and MEK.

Sensory and sympathetic innervation of the white fat tissue (WAT) contributes to lipolysis. In addition, both fiber types adapt in density to weight gain and loss. Because these findings are indicative for a tight control of nerve fiber plasticity by adipokines, we tested whether adipocytes control neurite growth of sensory neurons through angiopoietin-1 (Ang-1). We further considered initial hints that Ang-1-induced neuritogenesis involves transactivation of the high-affinity nerve growth factor (NGF) receptor trkA. Coculturing dorsal root ganglion (DRG) cells with 3T3-L1 adipocytes supported neurite outgrowth. These neurotrophic effects were associated with the increased expression of Ang-1 (presumably in adipocytes) as well as of trkA. The effects were abolished upon inactivating Ang-1 in culture with selective antibodies. Likewise, neurite outgrowth was impaired in the presence of inactivating NGF antibodies as well as upon inhibition of the NGF high-affinity trkA receptor with the antagonist K252a, indicating a tight cooperation of Ang-1 and NGF in the control of neuritogenesis. DRG-adipipocyte cocultures were further used to establish whether sensory neurons would form synaptic contacts with adipocytes. Electron microscopy demonstrated that cultured sensory neurons develop predominantly neuroneuronal synapses but seem to affect adipocytes by synapses en passant. Comparably to the case for neuritogenesis, expression of the presynaptic protein synaptophysin as well of the postsynaptic protein PSD-95 correlated with Ang-1 levels in culture. It is concluded that adipocyte-secreted Ang-1 supports neurite outgrowth, which is involved in synaptogenesis. The novel function of Ang-1 appears to play a physiological role in WAT plasticity., (Copyright 2006 Wiley-Liss, Inc.)

The human parasite Trypanosoma cruzi, the agent of Chagas' disease, expresses a membrane-bound neuraminidase/trans-sialidase, also known as parasite-derived neurotrophic factor, PDNF, because it binds and activates nerve growth factor (NGF) receptor TrkA in neuronal cells. Here, we identify a 21 amino acid region (425GNASQNVWEDAYRCVNASATAN445) of PDNF that reproduces its neurotrophic activities. Synthetic peptide Y21, modeled on this sequence, induces survival and neurite outgrowth in primary dorsal root ganglion neurons. Y21 but not other PDNF-based peptides promotes survival and neurite extension in TrkA-expressing but not in TrkA-deficient PC12 cells. Y21 also enhances phosphorylation of TrkA in PC12 cells and activation of Erk1/2 and Akt kinases with kinetics distinct from that of PDNF. In addition, Y21 stimulates phosphorylation of cAMP response element-binding protein, CREB. Peptide Y21, therefore, reproduces several TrkA-dependent activities of PDNF and NGF. However, Y21 inhibits the binding of PDNF but not NGF to TrkA. Similarly, Y21 blocks PDNF- but not NGF-dependent phosphorylation of Erk1/2. These findings raise the possibility that Y21 reacts with a TrkA site required for the binding of PDNF but not NGF. The functioning of Y21 as TrkA agonist reproducing TrkA-dependent biological activities of PDNF should help elucidate the mechanism of PDNF activation of TrkA-expressing cells and the design of small drugs for the treatment of Chagas' and other neurodegenerative diseases.

Nerve growth factor (NGF) binding to p75(NTR) influences TrkA signaling, yet the molecular mechanism is unknown. We observe that NGF stimulates TrkA polyubiquitination, which was attenuated in p75(-/-) mouse brain. TrkA is a substrate of tumor necrosis factor receptor-associated factor 6 (TRAF6), and expression of K63R mutant ubiquitin or an absence of TRAF6 abrogated TrkA polyubiquitination and internalization. NGF stimulated formation of a TrkA/p75(NTR) complex through the p62 scaffold, recruiting the E3/TRAF6 and E2/UbcH7. Peptide targeted to the TRAF6 binding site present in p62 blocked interaction with TRAF6 and inhibited ubiquitination of TrkA, signaling, internalization, and NGF-dependent neurite outgrowth. Mutation of K485 to R blocked TRAF6 and NGF-dependent polyubiquitination of TrkA, resulting in retention of the receptor on the membrane and an absence in activation of specific signaling pathways. These findings reveal that polyubiquitination serves as a common platform for the control of receptor internalization and signaling.

Metal ion homeostasis is regulated principally by metalloregulatory proteins that control metal ion uptake, storage and efflux genes. We have used transcriptional profiling to survey Bacillus subtilis for genes that are rapidly induced by exposure to high levels of metal ions including Ag(I), Cd(II), Cu(II), Ni(II) and Zn(II) and the metalloid As(V). Many of the genes affected by metal stress were controlled by known metalloregulatory proteins (Fur, MntR, PerR, ArsR and CueR). Additional metal-induced genes are regulated by two newly defined metal-sensing ArsR/SmtB family repressors: CzrA and AseR. CzrA represses the CadA efflux ATPase and the cation diffusion facilitator CzcD and this repression is alleviated by Zn(II), Cd(II), Co(II), Ni(II) and Cu. CadA is the major determinant for Cd(II) resistance, while CzcD protects the cell against elevated levels of Zn(II), Cu, Co(II) and Ni(II). AseR negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. Our results extend the range of identified effectors for the As(III)-sensor ArsR to include Cd(II) and Ag(I) and for the Cu-sensor CueR to include Ag(I) and, weakly, Cd(II) and Zn(II). In addition to systems dedicated to metal homeostasis, specific metal stresses also strongly induced pathways related to cysteine, histidine and arginine metabolism.

We studied whether nerve growth factor (NGF) can affect the membrane potential and conductance of PC12 cells. We demonstrate that NGF depolarizes the membrane of PC12 cells within a minute and by using transfected NIH 3T3-Trk and -p75 cells we show that both the high affinity NGF receptor p140(trk) and the low affinity NGF receptor or p75(NGF) may be involved in the depolarization. Tyrosine kinase inhibitor, K252a, partially inhibited the depolarization, but two agents affecting intracellular calcium movements, Xestospongin C (XeC) and thapsigargin, did not. The early depolarization was eliminated in Na+ free solutions and under this condition, a 'prolonged' (> 2 min) hyperpolarization was observed in PC12 cells in response to NGF. This hyperpolarization was also induced in PC12 cells by epidermal growth factor (EGF). Voltage clamp experiments showed that NGF produced a late (> 2 min) increase in membrane conductance. The Ca2+-dependent BK-type channel blocker, iberiotoxin, and the general Ca2+-dependent K+ channel blocker, TEA, attenuated or eliminated the hyperpolarization produced by NGF in sodium free media. Under pretreatment with the non-selective cation channel blockers La3+ and Gd3+, NGF hyperpolarized the membrane of PC12 cells. These results suggest that three different currents are implicated in rapid NGF-induced membrane voltage changes, namely an acutely activated Na+ current, Ca2+-dependent potassium currents and non-selective cation currents., (Copyright 2005 Wiley-Liss, Inc.)

The effects of vascular factors on the nervous system are still poorly investigated. Angiopoietin-1 (Ang-1), an endothelial cell growth factor with influences on blood vessel stabilization, has been recently reported to prevent apoptosis in a neuroblastoma cell line via a pathway dependent on Tie-2 receptor. The present study focuses on the effect of Ang-1 on cultured dorsal root ganglion (DRG) cells isolated from 1-day-old rats. Three-day-old DRG cultures were exposed to Ang-1 treatment under serum-free condition for another 5 days and stained with antibodies against neurofilament (NF) 200 protein. Neurite length and density increased compared with those of controls. Double-immunofluorescence staining demonstrated the co-localization of the Tie-2 receptor in some NF-200-positive perikarya. The reverse transcription/polymerase chain reaction technique identified Tie-2 receptor mRNA in intact DRG and in Ang-1-stimulated DRG cell cultures, but not in a Schwann cell line or in primary astrocyte cultures. Western blotting confirmed that the expression of NF 68 protein in cultures treated with Ang-1 or nerve growth factor was higher than that in cultures treated with medium alone. When the Tie-2 receptor was blocked with anti-Tie-2 receptor antibody, neurite outgrowth was severely impeded. Induction of trkA-receptor protein expression was observed to be dependent on the presence of Tie-2 receptors. We conclude that Ang-1 promotes neurite outgrowth from DRG cells positive for Tie-2 receptor. The signalling pathway appears to involve transactivation of the trkA receptor.

NGF binds to two receptors, p75NTR and TrkA. The endosomal trafficking of receptors is of emerging importance for the understanding of their signaling. We compared the endocytic trafficking of the two NGF receptors in PC12 cells. Both p75NTR and TrkA were internalized in response to NGF and colocalized with early endosomes. However, surprisingly, the subsequent endosomal trafficking paths of both NGF receptors diverged: whereas p75NTR recycled back to the surface, TrkA moved to late endosomes and underwent lysosomal degradation. By performing subcellular fractionations of NGF stimulated PC12 cells, tyrosine-phosphorylated TrkA was recovered in fractions corresponding to late endosomes. This implicates these organelles as novel endosomal NGF signaling platforms. Furthermore, the trafficking of NGF receptors could be manipulated by pharmacological means. Disrupting p75NTR recycling diminished TrkA activation in response to low concentrations of NGF, demonstrating a functional role for the recycling of p75NTR.

Connective tissue growth factor (CTGF) is implicated as a factor promoting tissue fibrosis in several disorders, including diabetic nephropathy. However, the molecular mechanism(s) by which it functions is not known. CTGF rapidly activates several intracellular signaling molecules in human mesangial cells (HMC), including extracellular signal-related kinase 1/2, Jun NH(2)-terminal kinase, protein kinase B, CaMK II, protein kinase Calpha, and protein kinase Cdelta, suggesting that it functions via a signaling receptor. Treating HMC with CTGF stimulated tyrosine phosphorylation of proteins 75 to 80 and 140 to 180 kD within 10 min, and Western blot analysis of anti-phosphotyrosine immunoprecipitates identified the neurotrophin receptor TrkA (molecular weight approximately 140 kD). Cross-linking rCTGF to cell surface proteins with 3,3'-dithiobis(sulfosuccinimidylpropionate) revealed that complexes formed with TrkA and with the general neurotrophin co-receptor p75(NTR). rCTGF stimulated phosphorylation of TrkA (tyr 490, 674/675). K252a, a known selective inhibitor of Trk, blocked this phosphorylation, CTGF-induced activation of signaling proteins, and CTGF-dependent induction of the transcription factor TGF-beta-inducible early gene in HMC. It is concluded that TrkA serves as a tyrosine kinase receptor for CTGF.

Targeted gene delivery to diseased subtypes of neurons will be beneficial to the success of gene therapy of neurological disorders. We designed a recombinant cationic polypeptide to facilitate gene delivery to neuronal-like PC12 cells that express the nerve growth factor (NGF) receptors. The recombinant polypeptide was composed of a targeting moiety derived from loop 4-containing hairpin motif of NGF and a DNA-binding moiety of 10-lysine sequence and expressed in Escherichia coli. It activated NGF receptor, TrkA and its downstream signaling pathways in PC12 and promoted the survival of neuronally differentiated PC12 cells deprived of serum. The polypeptide could also bind plasmid DNA and enhance polycation-mediated gene delivery in NGF receptor-expressing PC12 cells, but not in COS7 cells lacking NGF receptors. The enhancement of gene transfer in PC12 was inhibited by pretreatment of free, unbound polypeptides, suggesting a NGF-receptor-specific effect of the polypeptide. These observations demonstrated the concept of using receptor-mediated mechanism for targeted gene delivery to neurons.

The subcellular localization of transmembrane receptors and other signaling proteins has emerged as a key component in the regulation of the intensity and specificity of their activity. Recent research indicates that immature TrkA neurotrophin receptors are transactivated in the Golgi apparatus after stimulation of neuropeptide pituitary adenylate cyclase-activating polypeptide PAC1 receptors or adenosine A(2A) receptors. Transactivation occurs independently of the TrkA extracellular ligand, nerve growth factor (NGF), through a signaling pathway that is distinct from that used in the transactivation of other receptor tyrosine kinases and has consequences distinct from those elicited by NGF at the plasma membrane.

Neurotrophins activate a number of signaling pathways relevant to neuroprotection; however, their poor pharmacological properties and their pleiotropic effects resulting from interaction with the p75(NTR)-Trk-sortilin three-receptor signaling system limit therapeutic application. While local application of neurotrophin proteins addresses some of the pharmacological challenges, selective targeting of neurotrophin receptors might allow for more selective application of neurotrophin receptor signaling modulation. Recent studies have supported the feasibility of developing non-peptidyl small molecules that mimic specific domains of neurotrophins and modulate signaling of specific neurotrophin receptors. The expression of p75(NTR) by populations of neurons most vulnerable in Alzheimer's disease and the linkage of p75(NTR) signaling to aberrant signaling mechanisms occurring in this disorder, point to potential applications for p75(NTR)-based small molecule strategies. Small molecules targeted to p75(NTR) in the settings of neurodegenerative disease and other forms of neural injury might serve to inhibit death signaling, block proNGF-mediated degenerative signaling and minimize deleterious effects promoted by pharmacologically upregulated Trk signaling.

Melatonin is an endogenously generated potent antioxidant. Our previous studies indicate that melatonin improved learning and memory deficits in APP695 transgenic mouse of Alzheimer's disease. An ovariectomized (OVX) rat model which is characterized by progressive memory deficits, central cholinergic nerve system degeneration and differentiation/apoptosis imbalance is the ideal in vivo model in which to test the neuroprotective effects of melatonin. OVX Sprague-Dawley rats received daily injections of melatonin (5, 10 and 20 mg/kg) or 17 beta-estradiol (E2, 80 microg/kg) or sesame oil for 16 wk. Morris water maze results showed that ovarian steroid deprivation resulted in spatial memory impairment, while melatonin and E2 significantly ameliorated spatial memory deficits in OVX rats. The latency to find the hidden platform and the distance to reach the platform become shorter in both melatonin and E2-treated rats compared with those that were only OVX. Four months after OVX, the choline acetyltransferase activity in the frontal cortex and hippocampus were greatly decreased in comparison with the controls. Melatonin and E2 antagonized the effects induced by OVX. Interestingly, the activity of the acetylcholinesterase was not altered in any group of rats. DNA fragmentation was presented in the front cortex of the OVX rats. Melatonin and E2 reduced the number of apoptotic neurons. These findings demonstrate the important effects of melatonin and E2 on cholinergic neurons and support the potential application of melatonin in the treatment of dementia in postmenopausal women. Our results indicate that neuroprotection by melatonin partly correlated to modulation of apoptosis and protection of the cholinergic system. Early long-term melatonin application is a promising strategy which could potentially be applied in a clinic setting.

TrkA is a receptor tyrosine kinase activated primarily by nerve growth factor (NGF) to regulate differentiation, survival, and other important functions of neurons. Given the critical role TrkA plays in neural maintenance, it may be that microbial invaders of the nervous system utilize this receptor to reduce tissue damage for maximizing host-parasite equilibrium. Candidate pathogens could be those, like Trypanosoma cruzi, which may produce relatively little brain or nerve damage in long-lasting infections. We show here that T. cruzi, via its neuraminidase, binds TrkA in a NGF-inhibitable manner, induces TrkA autophosphorylation, and, through TrkA-dependent mechanisms, triggers phosphatidylinositol 3-kinase (PI3K)/Akt kinase signaling, cell survival, and neurite outgrowth. Unlike NGF, the neuraminidase does not react with the apoptosis-causing pan-neurotrophin receptor p75NTR. Therefore, these studies identify a novel and unique TrkA ligand in a microbial invader of the nervous system, raising the thus far unsuspected prospect of TrkA underlying clinical progression of an important human infectious disease.

Three new macrocyclic diterpenes, kansuinins F (1), G (2), and H (3), together with four known jatrophane diterpenes, kansuinins D (4), E (5), and A (6) and 3beta,5alpha,7beta,15beta-tetraacetoxy-9alpha-nicotinoyloxyjatropha-6(17)-11E-dien-14-one, were isolated from the roots of Euphorbia kansui. Compounds 1 and 2 were assigned as 6(17)-en-11,12-epoxy-14-one-type jatrophane diterpenes, and compound 3 as a 6(17)-en-11,14-epoxy-12-one jatrophane diterpene. The structures of compounds 1-3 and the relative configurations of compounds 4 and 5 were determined by spectral data analysis. Kansuinin E (5) exhibited a specific survival effect on fibroblasts that expressed TrkA, a high-affinity receptor for nerve growth factor.

Neurotrophins, such as NGF and BDNF, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and intracellular signaling. It has been shown that activation of Trk receptor tyrosine kinases can also occur via a G-protein-coupled receptor (GPCR) mechanism, without involvement of neurotrophins. Two GPCR ligands, adenosine and pituitary adenylate cyclase-activating polypeptide (PACAP), can activate Trk receptor activity to increase the survival of neural cells through stimulation of Akt activity. To investigate the mechanism of Trk receptor transactivation, we have examined the localization of Trk receptors in PC12 cells and primary neurons after treatment with adenosine agonists and PACAP. In contrast to neurotrophin treatment, Trk receptors were sensitive to transcriptional and translational inhibitors, and they were found predominantly in intracellular locations particularly associated with Golgi membranes. Biotinylation and immunostaining experiments confirm that most of the transactivated Trk receptors are found in intracellular membranes. These results indicate that there are alternative modes of activating Trk receptor tyrosine kinases in the absence of neurotrophin binding at the cell surface and that receptor signaling may occur and persist inside of neuronal cells.

Nerve growth factor (NGF) initially interested neurobiologists because of its effects in the developing nervous system. It is now clear that NGF functions throughout the life of the animal with a wide repertoire of actions. In the sensory nervous system it primarily influences the structure and function of nociceptors. Here, we provide a brief review of these actions and raise the overriding biological questions of why these multiple actions occur and how they are carried out.

Several studies have demonstrated the potential of olfactory ensheathing cells for the repair of central and peripheral nerve injury. However, the majority of these studies have been performed with olfactory ensheathing cells derived from the olfactory bulbs, situated inside the skull. A more clinically relevant source of olfactory ensheathing cells is the olfactory mucosa, located in the nose. To be successful, an autologous transplant of nasal ensheathing glia would require a large number of purified cells. To address this issue, we have focused our research on three neurotrophic factors, namely nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3). We show here that their respective receptors, TrkA, TrkB, TrkC, as well as p75(NTR) (the low affinity NGF receptor), are expressed in vitro by the nasal ensheathing cells; the three neurotrophins promote purification and proliferation of these glial cells, with an optimal concentration of 50 ng/ml; and human ensheathing cells can be easily biopsied and highly purified using a serum-free medium supplemented with NT3. This technique opens the door for clinical trials in which nasal ensheathing cells will be autotransplanted in humans suffering from nerve injury., (Copyright 2003 Wiley-Liss, Inc.)

Nerve growth factor (NGF) promotes the survival of embryonic sensory neurons and maintains the phenotypic characteristics of primary nociceptive neurons postnatally. NGF also contributes to nociceptor activation and hyperalgesia during inflammatory pain states. The purpose of this study was to determine whether NGF might have an additional pronociceptive action by interfering with opioid-mediated analgesia in primary nociceptive neurons. Sensory neurons were isolated from the dorsal root ganglia of weanling rats and kept in standard culture conditions either with or without exogenous NGF (50 ng/ml). Currents through voltage-gated calcium channels were recorded from individual neurons using the whole cell patch clamp technique with Ba(2+) as the charge carrier (I(Ba)). The micro-opioid agonist fentanyl (1 microM) and the GABA(B) agonist baclofen (50 microM) were used to test G protein-dependent inhibition of I(Ba). Fentanyl inhibited I(Ba) by an average of 38+/-4% in untreated cells vs. 25+/-2% in NGF-treated cells (P<0.01). NGF had no effect on I(Ba) current magnitude or kinetics. The NGF-induced attenuation of opioid action was observed as early as 4 h after exposure, but was not seen when NGF was applied by bath perfusion for up to 40 min, suggesting that the effect was not mediated by a rapid phosphorylation event. The effect of NGF was prevented by K-252a (100 nM), an inhibitor of TrkA autophosphorylation. Baclofen-induced inhibition of I(Ba), on the other hand, was not affected by NGF treatment, suggesting that NGF modulation of opioid-mediated inhibition occurred upstream from the G protein. This was supported by the finding that GTP-gamma-S, an agonist independent G protein activator, inhibited I(Ba) similarly in both untreated and NGF treated cells. The results show that NGF selectively attenuated opioid-mediated inhibition of I(Ba) via TrkA receptor activation, possibly by altering opioid receptor function.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that mediates synaptic plasticity and excitability in the CNS. Recent evidence has shown that increased BDNF levels can lead to hyperexcitability and epileptiform activities, while suppression of BDNF function in transgenic mice or by antagonist administration retards the development of seizures. However, several groups, including our own, have reported that increasing BDNF levels by continuous intrahippocampal infusion inhibits epileptogenesis. It is possible that the continuous administration of BDNF produces a down-regulation of its high-affinity TrkB receptor, leading to a decrease of neuronal responsiveness to BDNF. If so, then animals should respond differently to bolus injections of BDNF, which presumably do not alter Trk expression, compared with continuous infusion. To test this hypothesis, we compared the effects of intrahippocampal BDNF continuous infusion and bolus injections on kindling induction. We showed that continuous infusion of BDNF inhibited the development of behavioral seizures and decreased the level of phosphorylated Trks or TrkB receptors. In contrast, multiple bolus microinjections of BDNF accelerated kindling development and did not affect the level of phosphorylated Trks or TrkB receptors. Our results indicate that different administration protocols yield opposite effects of BDNF on neuronal excitability, epileptogenesis and Trk expression. Unlike nerve growth factor and neurotrophin-3, which affect mossy fiber sprouting, we found that BDNF administration had no effect on the mossy fiber system in naive or kindled rats. Such results suggest that the effects of BDNF on epileptogenesis are not modulated by its effect on sprouting, but rather by its effects on excitability.

PC12 cells undergo neuritogenesis upon nerve growth factor (NGF) activation of the TrkA receptor, an effect mimicked by the ganglioside GM1 binding B-subunit of cholera toxin (CTB). Modulation of neuritogenesis by a GM1 ligand indicates a possible pathway for pathophysiological actions of neuropathy-associated anti-GM1 antibodies. Here we examine the ability of GM1 binding toxins and antibodies to induce neuritogenesis, using a PC12 neurite outgrowth assay. Cholera toxin (CT) and commercially prepared CTB (sCTB, contaminated with traces of the adenyl cyclase activating CT A-subunit) were highly neuritogenic. Recombinant cholera toxin B-subunit (rCTB, free from CTA) induced a much smaller effect, suggesting that the potent effects of sCTB are largely due to contaminating CTA. The recombinant GM1 binding B-subunit of Escherichia coli heat-labile enterotoxin (rETxB) exhibited no neuritogenic activity, whilst rETx holotoxin, which activates adenyl cyclase, was highly neuritogenic. Monoclonal anti-GM1 IgM antibodies from human neuropathy subjects induced small neuritogenic effects. These data indicate that GM1/ligand interaction does not necessarily lead to neuritogenesis and suggest that a specialisation of CTB, not shared by anti-GM1 antibodies or rETxB, is required to activate TrkA. Our data also indicate that antibodies are unlikely to exert major modulatory effects on TrkA activity in patients with anti-GM1 antibody-associated peripheral neuropathies.

We report peripheral actions in rats of Neotrofin, a purine derivative of therapeutic interest. Systemic injections mimicked NGF in eliciting sprouting of nociceptive nerves without affecting their regeneration. The sprouting was prevented by anti-NGF treatment, implicating endogenous NGF. We detected no Neotrofin-induced increases in cutaneous NGF levels or in retrograde NGF transport. In contrast, both NGF and phosphorylation of trkA increased significantly in DRGs, with a marginal appearance of phosphorylated trkA in axons. We conclude that the DRG effects of Neotrofin are responsible for its induction of sprouting. Neotrofin also induced a striking phosphorylation of axonal erk 1 and 2, which was, however, unaffected by anti-NGF treatment. We suggest that this NGF-independent MAP kinase activation is involved in nonsprouting functions of Neotrofin such as neuroprotection. Unlike injected NGF, Neotrofin did not induce hyperalgesia, supporting its candidacy as a treatment for peripheral neuropathies like those induced by diabetes and anticancer chemotherapy.