Neutralization Of Nerve Growth Factor Research Articles

Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3

Orthodontic tooth movement elicits alveolar bone remodeling and orofacial pain that is manifested by tooth mechanical hyperalgesia. Nerve growth factor (NGF) is upregulated in periodontium and may modulate tooth mechanical hyperalgesia. The objectives were to examine the role of NGF in tooth mechanical hyperalgesia and to elucidate the underlying mechanisms. Tooth mechanical hyperalgesia was induced by ligating closed coil springs between incisors and molars in Sprague–Dawley rats. Retrograde labeling was performed by periodontal administration of fluor-conjugated NGF and the detection of fluorescence in trigeminal ganglia (TG). Lentivirus vectors carrying NGF shRNA were employed to knockdown the expression of NGF in TG. The administration of agonists, antagonists, and virus vectors into TG and periodontium was conducted. Tooth mechanical hyperalgesia was examined through the threshold of biting withdrawal. Our results revealed that tooth movement elicited tooth mechanical hyperalgesia that could be alleviated by NGF neutralizing antibody and that NGF was upregulated in periodontium (mainly in periodontal fibroblasts) and TG. Retrograde labeling revealed that periodontal NGF was retrogradely transported to TG after day 1. Acid-sensing ion channel 3 (ASIC3) and NGF were co-expressed in trigeminal neurons and the percentage of co-expression was significantly higher following tooth movement. The administration of NGF and NGF neutralizing antibody into TG could upregulate and downregulate the expression of ASIC3 in TG, respectively. NGF aggravated tooth mechanical hyperalgesia that could be alleviated by ASIC3 antagonist (APETx2). Moreover, NGF neutralizing antibody mitigated tooth mechanical hyperalgesia that could be recapitulated by ASIC3 agonist (GMQ). NGF-based gene therapy abolished tooth mechanical hyperalgesia and downregulated ASIC3 expression. Taken together, in response to force stimuli, periodontal fibroblasts upregulated the expressions of NGF that was retrogradely transported to TG, where NGF elicited tooth mechanical hyperalgesia through upregulating ASIC3. NGF-based gene therapy is a viable method in alleviating tooth-movement-induced mechanical hyperalgesia.

Nerve growth factor induced farnesoid X receptor upregulation modulates autophagy flux and protects hepatocytes in cholestatic livers

Upregulation of nerve growth factor (NGF) in parenchymal hepatocytes has been shown to exert hepatoprotective function during cholestatic liver injury. However, the modulatory role of NGF in regulation of liver autophagy remains unclear. This study aimed to scrutinize the regulatory role of NGF in hepatic expression of farnesoid X receptor (FXR), a bile acid (BA)-activated nuclear receptor, and to determine its cytoprotective effect on BA-induced autophagy and cytotoxicity. Livers of human hepatolithiasis and bile duct ligation (BDL)-induced mouse cholestasis were used for histopathological and molecular detection. The regulatory roles of NGF in autophagy flux and FXR expression, as well as its hepatoprotection against BA cytotoxicity were examined in cultured hepatocytes. FXR downregulation in human hepatolithiasis livers showed positive correlation with hepatic NGF levels. NGF administration upregulated hepatic FXR levels, while neutralization of NGF decreased FXR expression in BDL-induced cholestatic mouse livers. In vitro studies demonstrated that NGF upregulated FXR expression, increased cellular LC3 levels, and exerted hepatoprotective effect in cultured primary rat hepatocytes. Conversely, autophagy inhibition abrogated NGF-driven cytoprotection under BA exposure, suggesting involvement of NGF-modulated auophagy flux. Although FXR agonistic GW4064 stimulation did not affect auophagic LC3 levels, FXR activity inhibition significantly potentiated BA-induced cytotoxicity and increased cellular p62/SQSTM1 and Rab7 protein in SK-Hep1 hepatocytes. Moreover, FXR gene silencing abolished the protective effect of NGF under BA exposure. These findings support that NGF modulates autophagy flux via FXR upregulation and protects hepatocytes against BA-induced cytotoxicity. NGF/FXR axis is a novel therapeutic target for treatment of cholestatic liver diseases.

Inhibition of Nerve Growth Factor Signaling Alleviates Repeated Dural Stimulation-induced Hyperalgesia in Rats

Our previous study showed that acid-sensing ion channel 3 (ASIC3) in the trigeminal nucleus caudalis (TNC) is involved in the pathogenesis of recurrent migraine. ASIC3 is regulated by nerve growth factor (NGF), which induces hyperalgesia in various pain disorders. Neutralization of NGF is considered an effective treatment method. However, the contribution of NGF to repeated migraine-like attacks in chronic migraine (CM) remains unclear. Therefore, this study investigated the effect of NGF on ASIC3 expression in the TNC and the role of NGF signaling in chemical dural stimulation-induced hyperalgesia. A rat model was established by repeated dural infusions of inflammatory soup (IS) for seven days to simulate CM attacks. After repeated IS infusions, cutaneous hyperalgesia appeared in the rats’ periorbital region and hind paws, which showed significantly lower pain thresholds. IS infusions upregulated the mRNA and protein of NGF in the TNC, and NGF was mainly expressed in the cytoplasm of TNC neurons. An intracerebroventricular injection of an anti-NGF-neutralizing antibody relieved the cutaneous hyperalgesia of CM rats and decreased protein kinase C (PKC), ASIC3, calcitonin gene-related peptide (CGRP) and c-Fos expression in the TNC. Moreover, intracerebroventricular injection with the PKC blocker chelerythrine chloride alleviated IS infusion-induced hyperalgesia and reduced ASIC3, CGRP and c-Fos levels in the TNC. These results indicate that NGF might regulate ASIC3 expression via PKC activity in the TNC following repeated IS dural stimulation, and this signaling pathway might participate in IS-induced hyperalgesia.

Sustained pain hypersensitivity in the stressed colon: Role of mast cell‐derived nerve growth factor‐mediated enteric synaptic plasticity

Sustained pain hypersensitivity is the hallmark of stressed colon which could be partially explained by central sensitization with synaptic plasticity, the key mechanism of memory. We previously identified that synaptic plasticity of enteric nerve system (ENS) contributed to peripheral pain maintaining in the gut. However, the mechanisms of enteric "memory" formation remain elusive. In this study, rats were exposed to water avoidance stress (WAS) or sham stress (SS), with cromolyn sodium or physiological saline injected intraperitoneally 30minutes before stress every day. The abdominal withdrawal reflex scores, mesenteric afferent nerve activity, enteric neural c-fos expression, and enteric synaptic plasticity were assessed, and mast cell infiltration and degranulation. Furthermore, colonic mucosal mediators-induced enteric synaptic plasticity and the role of mast cell-derived nerve growth factor (NGF), tryptase, and histamine were investigated via ex vivo longitudinal muscle-myenteric plexus (LMMP) organotypic culture. It is shown that mast cell stabilizing inhibited WAS-induced visceral hypersensitivity through enhancing visceral pain threshold, decreasing spontaneous and distention-induced mesenteric afferent firing, and downregulating enteric neural activation (c-fos). Importantly, WAS led to evident enteric synaptic plasticity, but decreased by cromolyn. Water avoidance stress-derived mucosal supernatants markedly enhanced the c-fos expression and enteric synaptic plasticity in LMMP tissues, which could be eliminated by mast cell inhibition or NGF neutralization, but not tryptase or histamine blocking. In conclusion, mast cells/NGF pathway may be the key regulator of synaptic plasticity of ENS and participate in the formation of chronic stress-induced sustained visceral hypersensitivity.

Nerve growth factor-dependent hyperexcitability of capsaicin-sensitive bladder afferent neurones in mice with spinal cord injury.

What is the central question of this study? Nerve growth factor (NGF) is reportedly a mediator inducing urinary bladder dysfunction. Is NGF directly involved in hyperexcitability of capsaicin-sensitive C-fibre bladder afferent pathways after spinal cord injury (SCI)? What is the main finding and its importance? Neutralization of NGF by anti-NGF antibody treatment reversed the SCI-induced increase in the number of action potentials and the reduction in spike thresholds and A-type K+ current density in mouse capsaicin-sensitive bladder afferent neurones. Thus, NGF plays an important and direct role in hyperexcitability of capsaicin-sensitive C-fibre bladder afferent neurones attributable to the reduction in A-type K+ channel activity in SCI. Nerve growth factor (NGF) has been implicated as an important mediator in the induction of C-fibre bladder afferent hyperexcitability, which contributes to the emergence of neurogenic lower urinary tract dysfunction after spinal cord injury (SCI). In this study, we determined whether NGF immunoneutralization using an anti-NGF antibody (NGF-Ab) normalizes the SCI-induced changes in electrophysiological properties of capsaicin-sensitive C-fibre bladder afferent neurones in female C57BL/6 mice. The spinal cord was transected at the Th8/Th9 level. Two weeks later, continuous administration of NGF-Ab (10μgkg-1 h-1 , s.c. for 2weeks) was started. Bladder afferent neurones were labelled with Fast-Blue (FB), a fluorescent retrograde tracer, injected into the bladder wall 3weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurones were prepared. Whole-cell patch-clamp recordings were then performed in FB-labelled neurones. After recording action potentials or voltage-gated K+ currents, the sensitivity of each neurone to capsaicin was evaluated. In capsaicin-sensitive FB-labelled neurones, SCI significantly reduced the spike threshold and increased the number of action potentials during membrane depolarization for 800ms. These SCI-induced changes were reversed by NGF-Ab. Densities of slow-decaying A-type K+ (KA ) and sustained delayed rectifier-type K+ currents were significantly reduced by SCI. The NGF-Ab treatment reversed the SCI-induced reduction in the KA current density. These results indicate that NGF plays an important role in hyperexcitability of mouse capsaicin-sensitive C-fibre bladder afferent neurones attributable to a reduction in KA channel activity. Thus, NGF-targeting therapies could be effective for treatment of afferent hyperexcitability and neurogenic lower urinary tract dysfunction after SCI.

The effect of neutralization of nerve growth factor (NGF) on bladder and urethral dysfunction in mice with spinal cord injury.

To investigate the role of nerve growth factor (NGF) in lower urinary tract dysfunction in mice with spinal cord injury (SCI). Using 4-week SCI mice, single-filling cystometry and external urethral sphincter (EUS)-electromyography were performed under an awake condition. In some SCI mice, anti-NGF antibodies (10 µg/kg/h) were administered for 1 or 2 weeks before the urodynamic study. NGF levels in the bladder and L6/S1 spinal cord were assayed by ELISA. The transcript levels of P2X receptors and TRP channels in L6/S1 dorsal root ganglia (DRG) were measured by RT-PCR. In SCI mice, the area under the curve of non-voiding contractions (NVCs) during the storage phase was significantly decreased in both 1- and 2-week anti-NGF antibody-treated SCI groups. However, EUS-electromyogram parameters during voiding were not altered by the treatment. Bladder mucosal and spinal NGF levels were decreased after 2 weeks of anti-NGF antibody treatment. TRPA1 and TRPV1 transcripts in L6/S1 DRG were significantly decreased after 1- or 2-week anti-NGF treatment. In SCI mice, NGF is involved in the emergence of NVCs in association with increased expression of TRP receptors that are predominantly found in C-fiber afferent pathways. Thus, NGF targeting treatments could be effective for treating storage problems such as detrusor overactivity after SCI.

MP42-03 EFFECTS OF NERVE GROWTH FACTOR NEUTRALIZATION ON HYPEREXCITABILITY OF CAPSAICIN SENSITIVE BLADDER AFFERENT NEURONS IN MICE WITH SPINAL CORD INJURY

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2017MP42-03 EFFECTS OF NERVE GROWTH FACTOR NEUTRALIZATION ON HYPEREXCITABILITY OF CAPSAICIN SENSITIVE BLADDER AFFERENT NEURONS IN MICE WITH SPINAL CORD INJURY Takahiro Shimizu, Tsuyoshi Majima, Takahisa Suzuki, Nobutaka Shimizu, Naoki Wada, Shun Takai, Eiichiro Takaoka, Joonbeom Kwon, Pradeep Tyagi, Motoaki Saito, and Naoki Yoshimura Takahiro ShimizuTakahiro Shimizu More articles by this author , Tsuyoshi MajimaTsuyoshi Majima More articles by this author , Takahisa SuzukiTakahisa Suzuki More articles by this author , Nobutaka ShimizuNobutaka Shimizu More articles by this author , Naoki WadaNaoki Wada More articles by this author , Shun TakaiShun Takai More articles by this author , Eiichiro TakaokaEiichiro Takaoka More articles by this author , Joonbeom KwonJoonbeom Kwon More articles by this author , Pradeep TyagiPradeep Tyagi More articles by this author , Motoaki SaitoMotoaki Saito More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1292AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Nerve growth factor (NGF) has been implicated as an important mediator to induce C-fiber bladder afferent hyperexcitability, which contributes to the emergence of neurogenic detrusor overactivity (NDO) following spinal cord injury (SCI). In this study, we examined whether NGF neutralization using anti-NGF antibody normalizes the SCI-induced changes in electrophysiological properties of capsaicin-sensitive C-fiber bladder afferent neurons in the mouse model. METHODS In female C57BL/6 mice, the spinal cord was transected at the Th8/9 level. Two weeks later, an osmotic pump was placed subcutaneously to administer anti-NGF antibody at 10 µg/kg/h for 2 weeks. Bladder afferent neurons were labeled with axonal transport of Fast Blue (FB), a fluorescent retrograde tracer, injected into the bladder wall 3 weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurons were prepared. Whole cell patch clamp recordings were then performed in FB-labeled bladder afferent neurons, and the data were compared between SCI and spinal intact (SI) mice. After recording action potentials (AP) or voltage-gated K+ (Kv) currents, the sensitivity of each neuron to capsaicin was evaluated. RESULTS In capsaicin-sensitive bladder afferent neurons, the resting membrane potentials and the peak and duration of AP did not changed by SCI. On the other hand, the threshold for eliciting AP was significantly reduced in SCI vs. SI mice. Also, SCI increased the number of AP during 800 ms membrane depolarization. These SCI induced changes were reversed by NGF neutralization. SCI induced significant increases in the diameter and cell input capacitance of capsaicin-sensitive bladder afferent neurons, which were not reversed by NGF neutralization. Densities of slow decaying KA and sustained KDR currents evoked by depolarization to 0 mV were significantly reduced by SCI. NGF neutralization reversed the SCI-induced reduction in the KA current density. CONCLUSIONS In SCI mice, NGF plays an important role in hyperexcitability of capsaicin sensitive C-fiber bladder afferent neurons due to KA current reduction. Thus, NGF-targeting therapies could be effective for treatment of afferent hyperexcitability and NDO in SCI. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e544-e545 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Takahiro Shimizu More articles by this author Tsuyoshi Majima More articles by this author Takahisa Suzuki More articles by this author Nobutaka Shimizu More articles by this author Naoki Wada More articles by this author Shun Takai More articles by this author Eiichiro Takaoka More articles by this author Joonbeom Kwon More articles by this author Pradeep Tyagi More articles by this author Motoaki Saito More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Neutralization of nerve growth factor (NGF) inhibits the Th2 response and protects against the respiratory syncytial virus (RSV) infection.

Increasing evidence suggests that respiratory syncytial virus (RSV) infection during the early life is an important risk factor for the development of asthma. RSV infection is associated with neurogenic inflammation in the airways along with the increased expression of nerve growth factor (NGF). However, the role of NGF in RSV infection is not clear. In this study, we infected the rat with RSV and treated these animals with anti-NGF neutralization antibody. We found that anti-NGF treatment significantly alleviated the lung inflammation as evidenced by decreased inflammatory infiltration and decreased airway resistance. Importantly, anti-NGF treatment resulted in increased Th1, but decreased Th2 immune responses, and facilitated the viral control in the tissues and blood. Therefore, NGF inhibited Th2 but increased Th1 responses in RSV infection. Pharmacological intervention of NGF signaling during severe RSV infections could prevent or decrease further asthma symptoms.

Blocking Nerve Growth Factor Signaling Reduces the Neural Invasion Potential of Pancreatic Cancer Cells.

Perineural invasion (PNI) is thought to be one of the factors responsible for the high rate of tumor recurrence after surgery and the pain generation associated with pancreatic cancer. Signaling via the nerve growth factor (NGF) pathway between pancreatic cancer cells and the surrounding nerves has been implicated in PNI, and increased levels of these proteins have been correlated to poor prognosis. In this study, we examine the molecular mechanism of the NGF signaling pathway in PNI in pancreatic cancer. We show that knocking down NGF or its receptors, TRKA and p75NTR, or treatment with GW441756, a TRKA kinase inhibitor, reduces the proliferation and migration of pancreatic cancer cells in vitro. Furthermore, pancreatic cancer cells migrate towards dorsal root ganglia (DRG) in a co-culture assay, indicating a paracrine NGF signaling between the DRGs and pancreatic cancer cells. Knocking down the expression of NGF pathway proteins or inhibiting the activity of TRKA by GW441756 reduced the migratory ability of Mia PaCa2 towards the DRGs. Finally, blocking NGF signaling by NGF neutralizing antibodies or GW441756 inhibited the neurite formation in PC-12 cells in response to conditioned media from pancreatic cancer cells, indicating a reciprocal signaling pathway between the pancreatic cancer cells and nerves. Our results indicate that NGF signaling pathway provides a potential target for developing molecularly targeted therapies to decrease PNI and reduce pain generation. Since there are several TRKA antagonists currently in early clinical trials they could now be tested in the clinical situation of pancreatic cancer induced pain.

Bone marrow mesenchymal stem cells attenuate 2,5-hexanedione-induced neuronal apoptosis through a NGF/AKT-dependent pathway.

Growing evidence suggests that the increased neuronal apoptosis is involved in n-hexane-induced neuropathy. We have recently reported that bone marrow-mesenchymal stem cells-derived conditioned medium (BMSC-CM) attenuated 2,5-hexanedione (HD, the active metabolite of n-hexane)-induced apoptosis in PC12 cells. Here, we explored the anti-apoptotic efficacy of BMSC in vivo. HD-treated rats received BMSC by tail vein injection 5 weeks after HD intoxication. We found that in grafted rats, BMSC significantly attenuated HD-induced neuronal apoptosis in the spinal cord, which was associated with elevation of nerve growth factor (NGF). Neutralization of NGF in BMSC-CM blocked the protection against HD-induced apoptosis in VSC4.1 cells, suggesting that NGF is essential for BMSC-afforded anti-apoptosis. Mechanistically, we found that the decreased activation of Akt induced by HD was significantly recovered in the spinal cord by BMSC and in VSC4.1 cells by BMSC-CM in a TrkA-dependent manner, leading to dissociation of Bad/Bcl-xL complex in mitochondria and release of anti-apoptotic Bcl-xL. The importance of Akt was further corroborated by showing the reduced anti-apoptotic potency of BMSC in HD-intoxicated VSC4.1 cells in the presence of Akt inhibitor, MK-2206. Thus, our findings show that BMSC attenuated HD-induced neuronal apoptosis in vivo through a NGF/Akt-dependent manner, providing a novel solution against n-hexane-induced neurotoxicity.

Cartilage stem/progenitor cells are activated in osteoarthritis via interleukin-1β/nerve growth factor signaling.

IntroductionInterleukin-1β (IL-1β) and nerve growth factor (NGF) are key regulators in the pathogenesis of inflammatory arthritis; specifically, IL-1β is involved in tissue degeneration and NGF is involved in joint pain. However, the cellular and molecular interactions between IL-1β and NGF in articular cartilage are not known. Cartilage stem/progenitor cells (CSPCs) have recently been identified in osteoarthritic (OA) cartilage on the basis of their migratory properties. Here we hypothesize that IL-1β/NGF signaling is involved in OA cartilage degeneration by targeting CSPCs.MethodNGF and NGF receptor (NGFR: TrkA and p75NTR) expression in healthy and OA human articular cartilage and isolated chondrocytes was determined by immunostaining, qRT-PCR, flow cytometry and western blot. Articular cartilage derived stem/progenitor cells were collected and identified by stem/progenitor cell characteristics. 3D-cultured CSPC pellets and cartilage explants were treated with NGF and NGF neutralizing antibody, and extracellular matrix changes were examined by sulfated glycosaminoglycan (GAG) release and MMP expression and activity.ResultsExpression of NGF, TrkA and p75NTR was found to be elevated in human OA cartilage. Cellular changes upon IL-1β and/or NGF treatment were then examined. NGF mRNA and NGFR proteins levels were upregulated in cultured chondrocytes exposed to IL-1β. NGF was chemotactic for cells isolated from OA cartilage. Cells isolated on the basis of their chemotactic migration towards NGF demonstrated stem/progenitor cell characteristics, including colony-forming ability, multi-lineage differentiation potential, and stem cell surface markers. The effects of NGF perturbation in cartilage explants and 3D-cultured CSPCs were next analyzed. NGF treatment resulted in extracellular matrix catabolism indicated by increased sGAG release and MMP expression and activity; conversely, treatment with NGF neutralizing antibody inhibited increased MMP levels, and enhanced tissue inhibitor of matrix metalloprotease-1 (TIMP1) expression in OA cartilage explants. NGF blockade with neutralizing antibody also affected cartilage matrix remodeling in 3D-CSPC pellet cultures.ConclusionOur results strongly suggest that NGF signaling is a contributing factor in articular cartilage degeneration in OA, which likely targets a specific subpopulation of progenitor cells, the CSPCs, affecting their migratory and matrix remodeling activities. These findings provide novel cellular/signaling therapeutic targets in osteoarthritic cartilage.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0840-x) contains supplementary material, which is available to authorized users.

Nerve growth factor neutralization suppresses β-cell proliferation through activin A and betacellulin.

The aim of this study was to investigate the effects of nerve growth factor (NGF) neutralization on synthesis and secretion of activin A (Act-A) and betacellulin (BTC) from primary β cells and the importance of these relations for β-cell proliferation. β Cells were isolated from euglycemic and streptozotocin-induced (75 mg/kg) hyperglycemic rats and treated with NGF neutralization antibody. The gene expression levels of Act-A and BTC in the primary β cells were evaluated using quantitative real-time polymerase chain reaction. The cellular and secreted levels of Act-A and BTC proteins were estimated using Western blot analysis. Nerve growth factor neutralization (1) reduced β-cell proliferation, (2) decreased Act-A at gene expression and protein levels while increasing its secretion from β cells, and (3) increased BTC at gene expression level while mildly decreasing its cellular protein level and secretion from β cells. Nerve growth factor neutralization specifically affected β cells of hyperglycemic rats. These findings indicate that NGF is an important regulator for the synthesis and secretion of Act-A and BTC from the β cells. Moreover, the results suggested that β-cell proliferation decreased through NGF neutralization is possibly related to decreased BTC and increased Act-A secretion from β cells of hyperglycemic rats.

Neutralization of nerve growth factor impairs proliferation and differentiation of adult neural progenitors in the subventricular zone.

Adult neurogenesis is a multistep process regulated by several extrinsic factors, including neurotrophins. Among them, little is known about the role of nerve growth factor (NGF) in the neurogenic niches of the mouse. Here we analyzed the biology of adult neural stem cells (NSCs) from the subventricular zone (SVZ) of AD11 anti-NGF transgenic mice, in which the expression of the recombinant antibody aD11 leads to a chronic postnatal neutralization of endogenous NGF. We showed that AD11-NSCs proliferate 10-fold less, with respect to their control counterparts, and display a significant impairment in their ability to differentiate into β-tubulin positive neurons. We found a considerable reduction in the number of SVZ progenitors and neuroblasts also in vivo, which correlates with a lower number of newborn neurons in the olfactory bulbs of AD11 mice and a severe deficit in the ability of these mice to discriminate between different odors. We also demonstrated that, in AD11 mice, the morphology of both SVZ-resident and neurosphere-derived astrocytes is significantly altered. We were able to reproduce the AD11 phenotype in vitro, by acutely treating wild type NSCs with the anti-NGF antibody, further demonstrating that both the proliferation and the differentiation defects are due to the NGF deprivation. Consistently, the proliferative impairment of AD11 progenitors, as well as the atrophic morphology of AD11 astrocytes, can be partly rescued in vitro and in vivo by exogenous NGF addition. Altogether, our results demonstrate a causal link between NGF signaling and proper proliferation and differentiation of neural stem cells from the SVZ.

Ginger improves cognitive function via NGF-induced ERK/CREB activation in the hippocampus of the mouse

Ginger (the rhizome of Zingiber officinale Roscoe) has been used worldwide for many centuries in cooking and for treatment of several diseases. The main pharmacological properties of ginger include anti-inflammatory, antihyperglycemic, antiarthritic, antiemetic and neuroprotective actions. Recent studies demonstrated that ginger significantly enhances cognitive function in various cognitive disorders as well as in healthy brain. However, the biochemical mechanisms underlying the ginger-mediated enhancement of cognition have not yet been studied in normal or diseased brain. In the present study, we assessed the memory-enhancing effects of dried ginger extract (GE) in a model of scopolamine-induced memory deficits and in normal animals by performing a novel object recognition test. We found that GE administration significantly improved the ability of mice to recognize novel objects, indicating improvements in learning and memory. Furthermore, to elucidate the mechanisms of GE-mediated cognitive enhancement, we focused on nerve growth factor (NGF)-induced signaling pathways. NGF enzyme-linked immunosorbent assay analysis revealed that GE administration led to elevated NGF levels in both the mouse hippocampus and rat glioma C6 cells. GE administration also resulted in phosphorylation of extracellular-signal-regulated kinase (ERK) and cyclic AMP response element-binding protein (CREB), as revealed by Western blotting analysis. Neutralization of NGF with a specific NGF antibody inhibited GE-triggered activation of ERK and CREB in the hippocampus. Also, GE treatment significantly increased pre- and postsynaptic markers, synaptophysin and PSD-95, which are related to synapse formation in the brain. These data suggest that GE has a synaptogenic effect via NGF-induced ERK/CREB activation, resulting in memory enhancement.

In Vivo Regulation of Brain-Derived Neurotrophic Factor in Dorsal Root Ganglia Is Mediated by Nerve Growth Factor-Triggered Akt Activation during Cystitis

The role of brain-derived neurotrophic factor (BDNF) in sensory hypersensitivity has been suggested; however the molecular mechanisms and signal transduction that regulate BDNF expression in primary afferent neurons during visceral inflammation are not clear. Here we used a rat model of cystitis and found that the mRNA and protein levels of BDNF were increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. BDNF up-regulation in the L6 DRG was triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reduced BDNF levels during cystitis. The neutralizing NGF antibody also subsequently reduced cystitis-induced up-regulation of the serine/threonine kinase Akt activity in L6 DRG. To examine whether the NGF-induced Akt activation led to BDNF up-regulation in DRG in cystitis, we found that in cystitis the phospho-Akt immunoreactivity was co-localized with BDNF in L6 DRG, and prevention of the endogenous Akt activity in the L6 DRG by inhibition of phosphoinositide 3-kinase (PI3K) with a potent inhibitor LY294002 reversed cystitis-induced BDNF up-regulation. Further study showed that application of NGF to the nerve terminals of the ganglion-nerve two-compartmented preparation enhanced BDNF expression in the DRG neuronal soma; which was reduced by pre-treatment of the ganglia with the PI3K inhibitor LY294002 and wortmannin. These in vivo and in vitro experiments indicated that NGF played an important role in the activation of Akt and subsequent up-regulation of BDNF in the sensory neurons in visceral inflammation such as cystitis.

Quantitation of Chronic and Acute Treatment Effects on Neuronal Network Activity Using Image and Signal Analysis: Toward a High-Content Assay

Upon maturation, primary neuronal cultures form an interconnected network based on neurite outgrowth and synaptogenesis in which spontaneous electrical activity arises. Measurement of network activity allows quantification of neuronal health and maturation. A fluorescent indicator was used to monitor secondary calcium influxes after the occurrence of action potentials, allowing us to examine activity of hippocampal cultures via confocal live cell imaging. Subsequently, nuclear staining with DAPI allows accurate cell segmentation. To analyze the calcium recording in a robust, observer-independent manner, we implemented an automated image- and signal-processing algorithm and validated it against a visual, interactive procedure. Both methods yielded similar results on the emergence of synchronized activity and allowed robust quantitative measurement of acute and chronic modulation of drugs on network activity. Both the number of days in vitro (DIV) and neutralization of nerve growth factor (NGF) have a significant effect on synchronous burst frequency and correlation. Acute effects are demonstrated using 5-HT (serotonin) and ethylene glycol tetra-acetic acid. Automated analysis allowed measuring additional features, such as peak decay times and bursting frequency of individual neurons. Based on neuronal cell cultures in 96-well plates and accurate calcium recordings, the analysis method allows development of an integrated high-content screening assay. Because molecular biological techniques can be applied to assess the influence of genes on network activity, it is applicable for neurotoxicity or neurotrophics screening as well as development of in vitro disease models via, for example, pharmacologic manipulation or RNAi.

Affinity Crosslinking of Y1036 to Nerve Growth Factor Identifies Pharmacological Targeting Domain for Small Molecule Neurotrophin Antagonists

Classically, small molecule antagonists have targeted membrane bound receptors and intracellular enzyme targets. While this drug discovery strategy is extremely successful, the number of new chemical entities in the pharmaceutical pipeline is diminishing and complementary strategies are in need. A particularly attractive therapeutic strategy is to neutralize soluble signalling proteins using small molecules. Small molecule-based technologies have the potential to sufficiently alter the molecular topology of a given ligand and inhibit ligand/receptor interactions—effectively neutralizing the ligand’s signalling capacity. Recent technical advances in the field of structural biology have enabled the elucidation of ligand/receptor complexes at atomic resolution enabling a detailed appreciation of the molecular interactions governing ligand-mediated receptor activation. Exploiting molecular modeling techniques to study these signalling complexes allows for a paradigm shift from “receptorcentric” to “ligandcentric” screening strategies. Nerve growth factor (NGF) is a prototypical protein signalling ligand, which binds two receptors, TrkA and p75NTR. We first explore the molecular landscape governing the ligand/receptor interactions of NGF/TrkA and NGF/p75 structures. Next, we use the recently reported NGF neutralizing small-molecule, Y1036, as an affinity probe to determine residues in proximity to the pharmacological targeting domain of NGF and perform theoretical docking experiments to predict the residues which comprise distinct pharmacological targeting domains on the surface of NGF. Exploiting such strategies may facilitate “ligandcentric” drug discovery and could further the development of a trophic-factor-selective compound such as a BDNF-selective antagonist.

The relation among NGF, EGF and insulin is important for triggering pancreatic β cell apoptosis

Nerve growth factor (NGF) is a well-known mediator for maintaining the survival of neurons, while recent studies report that its absence induces apoptosis in cultured β cells of humans and rats. However, its relationship with other growth factors that have important roles in the survival and function of β cells such as epidermal growth factor (EGF) has not yet been elucidated. The aim of this study was to investigate the effects of NGF withdrawal on the synthesis and secretion of EGF, insulin with respect to β cell apoptosis in hyperglycemic rats. β cells were isolated from euglycemic and streptozotocin-induced hyperglycemic rats and treated with NGF neutralizing antibody for withdrawal of NGF in culture medium. NGF, EGF and insulin levels in cell lysates and secretion samples were measured by enzyme-linked immunosorbent assay, and their gene expressions were determined by real-time reverse transcription polymerase chain reaction assay. Apoptosis was quantitatively determined by cytoplasmic histone-associated DNA fragments. Nerve growth factor neutralization triggered β cell apoptosis. In addition decreased insulin, increased NGF and EGF were observed at gene expression and protein levels by NGF neutralization. Moreover, NGF withdrawal decreased secretion of these peptides from β cells. Although the alterations seemed to be similar under euglycemic and hyperglycemic conditions, NGF withdrawal more strongly affected β cells of hyperglycemic rats. These important findings indicate that NGF is an important regulator for the synthesis and secretion of EGF and insulin from the β cells. Moreover, results suggested that NGF withdrawal causes apoptosis by decreasing EGF, NGF and insulin secretion from β cells of hyperglycemic rats.

Role for NGF in augmented sympathetic nerve response to activation of mechanically and metabolically sensitive muscle afferents in rats with femoral artery occlusion

Arterial blood pressure and heart rate responses to static contraction of the hindlimb muscles are greater in rats whose femoral arteries were previously ligated than in control rats. Also, the prior findings demonstrate that nerve growth factor (NGF) is increased in sensory neurons-dorsal root ganglion (DRG) neurons of occluded rats. However, the role for endogenous NGF in engagement of the augmented sympathetic and pressor responses to stimulation of mechanically and/or metabolically sensitive muscle afferent nerves during static contraction after femoral artery ligation has not been specifically determined. In the present study, both afferent nerves and either of them were activated by muscle contraction, passive tendon stretch, and arterial injection of lactic acid into the hindlimb muscles. Data showed that femoral occlusion-augmented blood pressure response to contraction was significantly attenuated by a prior administration of the NGF antibody (NGF-Ab) into the hindlimb muscles. The effects of NGF neutralization were not seen when the sympathetic nerve and pressor responses were evoked by stimulation of mechanically sensitive muscle afferent nerves with tendon stretch in occluded rats. In addition, chemically sensitive muscle afferent nerves were stimulated by lactic acid injected into arterial blood supply of the hindlimb muscles after the prior NGF-Ab, demonstrating that the reflex muscle responses to lactic acid were significantly attenuated. The results of this study further showed that NGF-Ab attenuated an increase in acid-sensing ion channel subtype 3 (ASIC3) of DRG in occluded rats. Moreover, immunohistochemistry was employed to examine the number of C-fiber and A-fiber DRG neurons. The data showed that distribution of DRG neurons with different thin fiber phenotypes was not notably altered when NGF was infused into the hindlimb muscles. However, NGF increased expression of ASIC3 in DRG neurons with C-fiber but not A-fiber. Overall, these data suggest that 1) NGF is amplified in sensory nerves of occluded rats and contributes to augmented reflex sympathetic and blood pressure responses evoked by stimulation of chemically, but not mechanically, sensitive muscle afferent nerves and 2) NGF likely plays a role in modulating the muscle metaboreflex via enhancement of ASIC3 expression in C-fiber of DRG neurons.

Regulation of IGF-1 but not TGF-β1 by NGF in the smooth muscle of the inflamed urinary bladder

Intraperitoneal injection of cyclophosphamide (CYP) causes hemorrhagic cystitis with excess growth of muscular layer leading to bladder hypertrophy; this could be attributable to changes in the expression profiles of growth factors in the inflamed urinary bladder. The growth factors characterized in the current study include nerve growth factor (NGF), insulin-like growth factor (IGF)-1, and transforming growth factor (TGF)-β1. We found that following CYP injection for 8h and 48h, the mRNA levels of all three factors were increased in the inflamed bladder when compared to control. The level of NGF mRNA was mainly increased in the urothelium layer while the levels of IGF-1 mRNA and TGF-β1 mRNA were increased in the smooth muscle layer. The level of NGF high affinity receptor TrkA mRNA was also increased in both the urothelium and the smooth muscle layers during bladder inflammation. When we blocked NGF action with NGF neutralizing antibody in vivo, we found that the up-regulation of IGF-1 in the inflamed bladder was reversed while the up-regulation of TGF-β1 was not affected by NGF neutralization. The effect of NGF on regulating IGF-1 expression was further confirmed in bladder smooth muscle culture showing that exogenous NGF increased the mRNA level of IGF-1 after 30min to 1h stimulation. These results suggested that bladder inflammation induced region-specific changes in the expression profiles of NGF, IGF-1 and TGF-β1. The up-regulation of NGF in the urothelium may have a role in affecting bladder smooth muscle cell physiology by regulating IGF-1 expression.