Source Of Nerve Growth Factor Research Articles

MP03-04 SECRETION OF NEUROTROPHINS AND THEIR PRECURSORS IS UNDER CONTROL OF NATRIURETIC PEPTIDE A IN BLADDER CELLS IN CULTURE

You have accessJournal of UrologyCME1 Apr 2023MP03-04 SECRETION OF NEUROTROPHINS AND THEIR PRECURSORS IS UNDER CONTROL OF NATRIURETIC PEPTIDE A IN BLADDER CELLS IN CULTURE Claudia Covarrubias, Philippe Cammisotto, and Lysanne Campeau Claudia CovarrubiasClaudia Covarrubias More articles by this author , Philippe CammisottoPhilippe Cammisotto More articles by this author , and Lysanne CampeauLysanne Campeau More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urine storage and bladder voiding are under control of the peripheral and central nervous systems. Neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), controls neuroregeneration while their respective precursor proNGF and proBDNF trigger inflammatory processes and apoptosis. The ratio between neurotrophins to proneurotrophins must therefore be tighly controlled and constitutes a viable biomarker for overactive bladder syndrome (OAB). On the other hand, natriuretic peptide A (ANP) that is increased during aging, has been linked to OAB. We here examine how ANP is involved in the secretion of neurotrophins by cells of the bladder. METHODS: Smooth muscle (SMC) and urothelial cells (URO) were grown from rat bladder after collagenase digestion. After confluency, they were incubated for 24 hours with ANP (100 nM). NGF, proNGF, BDNF, proBDNF and matrix metalloproteinase-9 (MMP-9) were measured using specific kits. RESULTS: We found that both cell types are major source of NGF and proNGF while BDNF and proBDNF were mainly released by SMC. ANP decreases NGF and proBDNF secretion by SMC, while proNGF and BDNF levels were not affected, mimicking the observations obtained in urine samples of a female cohort of patients diagnosed with OAB. Interestingly, ANP also increased MMP-9 activity in the same cell culture medium. In UROs, no changes in the secretion of neurotrophins or proneurotrophins could be observed, however, the secretion of active MMP-9 was found potently enhanced by ANP with final levels around 20 times higher than the ones found in SMC medium. Finally, using CrisprCas9 to knockdown MMP-9 gene, we confirmed the essential role of this enzyme in the proteolysis of NGF into peptides and in the conversion of proBDNF to BDNF. CONCLUSIONS: Bladder cells exhibit unique pattern of neurotrophin secretions. Both are affected by ANP, SMC mostly at the level of neurotrophin secretions and urothelial cells in the amount of MMP-9 released in the extracellular medium. We suggest that the imbalance in neutrophin ratios observed in the urine of patients with OAB could result, at least in part, from a direct action of ANP on bladder cells. Source of Funding: Canadian Urologist Association and Quebec Network for Research on Aging (RQRV) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e22 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Claudia Covarrubias More articles by this author Philippe Cammisotto More articles by this author Lysanne Campeau More articles by this author Expand All Advertisement PDF downloadLoading ...

Localization of NGF expression in mouse spleen and salivary gland: Relevance to pleotropic functions

Our primary goal was to determine if leukocytes are a source of nerve growth factor (NGF) in mouse spleen. Noradrenergic nerves were localized to arteries and white pulp in normal spleens but only to arteries in ultra-immunodeficient R2G2 mice that lack leukocytes. NGF mRNA was detected in vascular cells and leukocytes of normal spleen, and several of the latter were T cells based on double labeling for NGF mRNA and CD3. Our findings indicate NGF is produced by vascular cells and to a lesser extent by leukocytes in spleen and provide support for pleiotropic actions in spleen and salivary glands.

PD27-12 ANTAGONISM OF THE P75NTR RECEPTOR INCREASES THE RELEASE OF NERVE GROWTH FACTOR IN UROTHELIAL CELLS BY INHIBITING MATRIX METALLOPROTEINASE-9

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology (PD27)1 Sep 2021PD27-12 ANTAGONISM OF THE P75NTR RECEPTOR INCREASES THE RELEASE OF NERVE GROWTH FACTOR IN UROTHELIAL CELLS BY INHIBITING MATRIX METALLOPROTEINASE-9 Aalya Hamouda, Philippe Cammisotto, Stephanie Sirmakesyan, Uri Saragovi, and Lysanne Campeau Aalya HamoudaAalya Hamouda More articles by this author , Philippe CammisottoPhilippe Cammisotto More articles by this author , Stephanie SirmakesyanStephanie Sirmakesyan More articles by this author , Uri SaragoviUri Saragovi More articles by this author , and Lysanne CampeauLysanne Campeau More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002020.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Decreased levels of nerve growth factor (NGF) have been observed in the urine of patients with overactive bladder syndrome (OAB) and of diabetic animal models. This unbalance appeared to result from an increased activity of the proteolytic enzyme metalloproteinase-9 (MMP-9). On the other hand, THX-B, an inhibitor of p75NTR, restored normal levels of NGF in mice with type 1 diabetes. We here examine in vitro the effect of THX-B on the activity of MMP-9 in bladder cells. METHODS: Primary culture of urothelial and smooth muscle cells (SMCs) were grown from mouse bladder and exposed to p75NTR antagonist THX. The expression of NGF and MMP-9 were assessed by RT-qPCR, by immunohistochemistry and by immunoblotting. Levels of microRNAs were measured by RT-qPCR. NGF and proNGF secretion were measured by ELISA kits and MMP-9 activity by enzymatic assays. RESULTS: The mRNAs for NGF and MMP-9 were detected and found expressed in both cell types at similar level. Microscopy confirmed the localization of both proteins in cells. Urothelial cells and SMCs were both major source of NGF and proNGF. On the other hand, MMP-9 protein content was 7 times higher in SMCs than in urothelial cells, which was confirmed by high levels of miR-491-5p in the latter. However, secretion of active MMP-9 in the medium was 40 times higher in urothelial cells medium. Incubation with THX-B (5 µg/mL) for 24 hours abolished the synthesis and secretion of MMP-9 and doubled the concentration of NGF in the medium of urothelial cells. ProNGF secretion levels were not affected. THX-B had little effects on SMCs both at the level of NGF and MMP-9. CONCLUSIONS: Urothelial cells appear to be the primary target for THX-B to modulate secretion of NGF and MMP-9. These results are in accordance with our previous publications on OAB patients and diabetes type 1 in rodents, and suggest that THX-B could be a therapeutic tool to improve OAB by targeting primarily the urothelium. Source of Funding: Canadian Urology AssociationQuebec Network for Research on Aging © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e448-e448 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Aalya Hamouda More articles by this author Philippe Cammisotto More articles by this author Stephanie Sirmakesyan More articles by this author Uri Saragovi More articles by this author Lysanne Campeau More articles by this author Expand All Advertisement Loading ...

Abstract 7: Nerve Growth Factor Derives From Pericytes And Smooth Muscle Cells After Extremity Trauma

Purpose: Neurotrophic factors like nerve growth factor (NGF) have been described in the literature as a crucial regulator in developmental biology and inflammation of musculoskeletal tissues. While extremity trauma has been shown to elicit re-engagement of developmental programs like NGF upregulation and invasion of new sensory nerve endings in a mature organism, the source of NGF has remained largely unknown. In a validated extremity trauma mouse model, we have previously demonstrated a significant attenuation of injury site re-innervation by sensory fibers following inhibiting of afferent neural signaling and NGF binding of its receptor TrkA. Given this significant impact of NGF on sensory reinnervation of injured soft tissues and downstream bone formation, we examined the source of NGF after extremity trauma using single cell transcriptomic and reporter proteomic technologies. Methods: A 30% dorsal burn and Achilles transection was performed. The tendon site tissues were harvested from baseline (t0) and day 3, 7, and 21 (n=3-4/group) after induction. Samples were prepared for library generation on a 10x Genomics Chromium Controller, sequenced on a Illumina HiSeq 4000, and analyzed with Cell Ranger Software for pre-processing and alignment to the mm10 genome. Downstream analyses including unsupervised clustering and canonical correlation analyses were performed with Seurat. Immunofluorescent (IF) labeling of cellular markers including αSMA was performed using NGF-eGFP reporter mice at baseline, and at 1,3 and 9 weeks after injury (n=2-3/group). Results: To localize cell specific Ngf expression from injured soft tissue, 9 cell clusters were defined across all timepoints: mesodermal (Prrx1) populations including Acta2+ pericyte/vascular smooth muscle (SMC) and Pdgfra+ mesenchymal cells, two Pecam1+ endothelium, and four inflammatory cell populations (mixed, B cell, T cell, and neutrophil). Ngf was found uniquely enriched in the pericyte/SMC cluster in a composite view. These pericytes/SMCs were found in increasing number and Ngf expression across timepoints, peaking at day 21. To further characterize this joint cluster (1273 cells), the pericyte/SMC cluster was isolated and blindly re-clustered to produce new sub-clusters, distinguishing Pdgfrb (platelet derived growth factor receptor beta)highPrrx1highAbcc9+ pericytes from Acta2highPln+Myh11high SMCs; an uncharacterized cluster with 24 cells was discarded from analysis. Of these two sub-clusters, vascular SMCs demonstrated the highest Ngf expression at baseline (mean normalized Ngf expression of 0.52 [in 41% of cells] vs 0.28 [in 23% of cells]. IF of NGF-eGFP reporter mice show robust NGF co-localization with αSMA+ SMCs. Conclusions: This is the first work characterizing the pericyte and vascular SMC as a major contributor to NGF signaling at an extremity injury site. The fine resolution defining the cellular source of NGF provides insight into potential mechanisms correlating nascent nerve and vascular growth given the intersection of these pathways at the level of pericytes and myofibroblastic SMCs that will inform future candidate therapeutics to improve extremity trauma healing and re-innervation.

Murine and Chinese cobra venom‑derived nerve growth factor stimulate chondrogenic differentiation of BMSCs in vitro: A comparative study.

Mesenchymal stem cell (MSC)-based therapy has been commonly used in cartilage reconstruction, due to its self-renewing ability and multi-differentiation potential. Nerve growth factor (NGF) from cobra venom has been reported to regulate chondrogenesis of bone-derived MSCs (BMSCs) and chondrocyte metabolism. Therefore, the present study aimed to determine whether other sources of NGF behave in the same manner as NGF from natural venom. The present study compared the effects of NGF from two sources, the commercially purchased recombinant murine β-NGF (mNGF) and cobra venom-derived NGF (cvNGF), on chondrogenesis of BMSCs by performing hematoxylin and eosin and fluorescein diacetate/propidium iodide staining, DNA and glycosaminoglycan quantization and reverse transcription-quantitative polymerase chain reaction to investigate cell morphology, viability, proliferation, glycosaminoglycan synthesis and cartilage-specific gene expression. The results demonstrated that cvNGF significantly accelerated cell proliferation and upregulated the expression of cartilage-specific genes, including aggrecan, SRY-box 9 and collagen type II α1 chain. Conversely, cvNGF reduced the expression levels of collagen type I α1 chain (a fibrocartilage marker), runt-related transcription factor 2 and enolase 2 compared with in the mNGF and control groups. In addition, Chinese cobra venom, which is the main resource of cvNGF, is abundant and inexpensive, thus greatly decreasing the cost. In conclusion, the present study demonstrated that cvNGF may be considered a potential growth factor for inducing chondrogenic differentiation of BMSCs.

Butyrate promotes visceral hypersensitivity in an IBS-like model via enteric glial cell-derived nerve growth factor.

Altered visceral sensation is common in irritable bowel syndrome (IBS) and nerve growth factor (NGF) participates in visceral pain development. Sodium butyrate (NaB) could induce colonic hypersensitivity via peripheral up-regulation of NGF in animals. Enteric glial cells (EGCs) appear to be an important source of NGF. Whether butyrate could induce visceral hypersensitivity via increased EGC-derived NGF is still unknown. CRL-2690 cells were used for transcriptome analyses after butyrate treatment. Rats received butyrate enemas to induce colonic hypersensitivity. Colorectal distention test was performed to assess visceral sensitivity. Immunofluorescence studies were used to evaluate the co-expression of glial fibrillary acidic protein (GFAP) and NGF or growth associated protein 43 in animal model. NGF expression in rat colon was also investigated. In vitro, CRL-2690 cells were stimulated with NaB or trichostatin A (TSA). NGF or GFAP expression was also examined. Transcriptome analyses showed that butyrate induced marked changes of genes expression related to neurotrophic signaling pathways. NaB-treated rats showed increased visceral sensitivity. An improved NGF expression level was observed in NaB-treated rats. Meanwhile, a 2.1-fold increase in co-expression of GFAP and NGF was also determined in rats received NaB enemas. In cultured cells, both NaB and TSA treatment could cause obvious NGF expression. Thus, butyrate might regulate EGC function via histone deacetylase inhibition. Butyrate-EGC interplay may play a pivotal role in regulation of NGF expression and the development of colonic hypersensitivity in IBS-like animal model.

NGF-dependent axon growth and regeneration are altered in sympathetic neurons of dystrophic mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal disease, determined by lack of dystrophin (Dp427), a muscular cytoskeletal protein also expressed by selected neuronal populations. Consequently, besides muscular wasting, both human patients and DMD animal models suffer several neural disorders. In previous studies on the superior cervical ganglion (SCG) of wild type and dystrophic mdx mice (Lombardi et al. 2008), we hypothesized that Dp427 could play some role in NGF-dependent axonal growth, both during development and adulthood. To address this issue, we first analyzed axon regeneration potentials of SCG neurons of both genotypes after axotomy in vivo. While noradrenergic innervation of mdx mouse submandibular gland, main source of nerve growth factor (NGF), recovered similarly to wild type, iris innervation (muscular target) never did. We, therefore, evaluated whether dystrophic SCG neurons were poorly responsive to NGF, especially at low concentration. Following in vitro axotomy in the presence of either 10 or 50ng/ml NGF, the number of regenerated axons in mdx mouse neuron cultures was indeed reduced, compared to wild type, at the lower concentration. Neurite growth parameters (i.e. number, length), growth cone dynamics and NGF/TrkA receptor signaling in differentiating neurons (not injured) were also significantly reduced when cultured with 10ng/ml NGF, but also with higher NGF concentrations. In conclusion, we propose a role for Dp427 in NGF-dependent cytoskeletal dynamics associated to growth cone advancement, possibly through indirect stabilization of TrkA receptors. Considering NGF activity in nervous system development/remodeling, this aspect could concur in some of the described DMD-associated neural dysfunctions.

Myenteric expression of nerve growth factor and the p75 neurotrophin receptor regulate axonal remodeling as a consequence of colonic inflammation in mice

Nerve growth factor (NGF) levels increase in response to inflammation of the mammalian colon. The precise cellular sources of colonic NGF synthesis, however, remain elusive. Using lines of transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the NGF promoter, we found a subpopulation of adendritic EGFP+ neurons in the myenteric plexus. These colonic EGFP+ neurons display positive immunostaining for calretinin but not nitric oxide synthase 1 (NOS1) two biomarkers of mouse myenteric neurons. A loss of NGF expression in null mutant postnatal mice does not affect the survival of these EGFP+ neurons. Induction of colonic inflammation confirms local increases in NGF mRNA/protein levels, which coincide with heightened detection of EGFP by myenteric neurons. Though NOS1+ myenteric neurons display positive immunostaining for trkA (the receptor required for NGF binding/signaling), transgenic overexpression of NGF by smooth muscle cells in the colon does not alter the survival, somal size, or axonal density of trkA-expressing NOS1+ myenteric neurons. Mice lacking functional p75NTR (the second receptor required for NGF binding) exhibit significantly less axonal damage among NOS1+ myenteric neurons, in response to chemically induced colonic inflammation. Likewise, trkA-expressing sympathetic axons that innervate the myenteric ganglia display less damage in the absence of p75NTR. These data are the first to implicate calretinin+ myenteric neurons as a source of NGF in the murine colon, and that in response to colonic inflammation, increases in NGF can exaggerate damage of intrinsic NOS1+ axons and extrinsic sympathetic axons that co-express trkA and p75NTR.

TGF-β is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5-Smad2/3 pathway. Potential role in OA related pain?

Pain is the main problem for patients with osteoarthritis (OA). Pain is linked to inflammation, but in OA a subset of patients suffers from pain without inflammation, indicating an alternative source of pain. Nerve Growth Factor (NGF) inhibition is very efficient in blocking pain during OA, but the source of NGF is unclear. We hypothesize that damaged cartilage in OA releases Transforming Growth Factor-β (TGF-β), which in turn stimulates chondrocytes to produce NGF. Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-β1 and/or Interleukin-1 (IL-1)β. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1, Smad2/3 or Smad1/5/8 signaling to identify the TGF-β pathway inducing NGF. NGF expression was consistently induced in higher levels by TGF-β than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. TAK1 inhibition consistently reduced TGF-β-induced NGF whereas it fully blocked IL-1β-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-β-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-β exposure led to a consistent high level of NGF induction. We show for the first time that TGF-β induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA.

Induction of salivary nerve growth factor by Yogic breathing: a randomized controlled trial

Nerve growth factor (NGF), a trophic factor involved in the development, maintenance, and survival of the peripheral nervous system and the cholinergic neurons of the central nervous system, is significantly reduced in Alzheimer's Disease (AD) patients (Aloe et al., 2012). Particularly basal forebrain complex (BFC) neurons are highly affected in AD patients. NGF protects BFC neurons in experimental trauma models and in age-associated cholinergic decline. Therefore, administration of NGF is investigated as a therapeutic option for AD patients (Aloe et al., 2012). NGF therapy is limited by hyperalgesia, limited bioavailability, and use of invasive methods including intracranial injection of adeno-associated virus-based gene delivery vector, which constitutively expresses human NGF. In this context, non-pharmacological modes of treatment such as meditation and Yoga have been considered as alternative approaches to treat neurological disorders. Yogic breathing (YB, also called Pranayama) is a collection of techniques to regulate breathing voluntarily. YB induces a strong relaxation response via vagal and parasympathetic stimulation (Jerath et al., 2006). Such responses are causally linked to rapid changes in gene expression, particularly to those genes controlling stress, inflammation, and metabolism. Saliva is a known source of NGF, and incidentally, Yoga and relaxation stimulate salivation. Saliva (0.75–1.5 L/day in humans) contains numerous biologically active molecules to be potentially useful as diagnostic markers and as therapeutic clues. Salivary secretion regulates the digestive, nervous, immune, and respiratory systems. We conducted a pilot study to investigate whether YB could potentially stimulate salivary expression of NGF in cognitively normal healthy volunteers. This could serve as a model for future studies in AD patients.

Intermittent vs Continuous Administration of Nerve Growth Factor to Injured Medial Septal Cholinergic Neurons in Rat Basal Forebrain

Many medial septal neurons of the basal forebrain are dependent on nerve growth factor (NGF) from the hippocampus for survival and maintenance of a cholinergic phenotype. When deprived of their source of NGF by axotomy, medial septal neuronal cell bodies atrophy and lose their cholinergic markers. This is similar to what is observed in the basal forebrain during Alzheimer’s disease (AD). In the present study, medial septal neurons were axotomized in female rats by way of a fimbria/fornix lesion. For fourteen days following axotomy, varying NGF doses (1 – 250 μg/ml) were administered to the lateral cerebral ventricle with either mini-osmotic infusion or daily injection. The responsiveness of medial septal neurons was evaluated with choline acetyltransferase immunohistochemistry. Within the mini-osmotic pumps, NGF activity diminished greatly during the first five days of implantation, but increased dramatically in the CSF after five days of infusion. The responsiveness of medial septal neurons to NGF was dose dependent and the ED50 for NGF injection was determined to be 14.08 μg/ml compared to 27.60 μg/ml for NGF infusion. Intermittent injections at varying intervals were evaluated with 30 μg/ml NGF over a fourteen-day time period (2, 3, 6, or 12 injections). No differences occurred in the number of choline acetyltransferase neurons from rats that received weekly injections to those that received daily injections of NGF. NGF administration has been suggested as a therapy for AD. The results of these studies continue to highlight the need for NGF stability within the delivery system and AD patient CSF, the choice of delivery system, frequency of administration, and the NGF dose for maintaining basal forebrain cholinergic neurons during AD.

Homage to Rita Levi‐Montalcini, the queen of modern neuroscience

The first cell growth factor, nerve growth factor (NGF), was discovered by Rita Levi-Montalcini (RLM) in the early 1950s. Originally identified as neurite outgrowth-stimulating factor, later studies revealed that non-neuronal cells, including immune cells, endothelial cells, cardiomyocytes, pancreatic beta cells, prostate epithelial and adipose tissue cells, were also targets for and/or sources of NGF. Nerve growth factor is well recognised as mediating multiple biological phenomena, ranging from the neurotrophic through immunotrophic and epitheliotrophic to metabotrophic effects. Consequently, NGF and other members of the neurotrophin family are implicated in the pathogenesis of a large spectrum of neuronal and non-neuronal diseases, ranging from Alzheimer's and other neurodegenerative diseases to atherosclerosis and cardiometabolic disorders. Recent studies have demonstrated the therapeutic potentials of NGF in these conditions, including ocular and cutaneous diseases. NGF TrkA receptor antagonists emerged as novel drugs for pain, prostate and breast cancer, melanoma and urinary bladder syndromes. Here, we briefly describe the 'unpredictable' ideogenesis of the discovery of NGF, a eureka in the neuroscience.

Myenteric Dogiel II Neurons Express Nerve Growth Factor

We have generated lines of transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the murine nerve growth factor (NGF) promoter. These mice afford us unique temporal and spatial insight into those cellular populations that display NGF promoter activity from early mammalian embryogenesis to adulthood. These mice also offer us the opportunity to assess those cells that express NGF promoter activity in response to injury and/or disease. We have made particular note of a population of EGFP‐positive cells in the colon of these transgenic mice. In adult animals, these EGFP‐positive neurons can be unequivocally identified as a population residing within the myenteric plexus. These neurons have large smooth cell bodies, with two or three axonal processes that give rise to a dense plexus of fibers within the enteric ganglia (these axonal plexuses are also visualized by EGFP). Based on these anatomical features, we have speculated that these EGFP‐positive neurons are primary afferent (or Dogiel type II) neurons. Preliminary assessments of the neurochemical features of these EGFP‐positive neurons reveal that most express the calcium‐binding protein calretinin. We will present additional data regarding the localization of other biomarkers for Dogiel type II neurons, including calcitonin gene‐related peptide. We will also show how these EGFP‐positive cells react to chemically‐induced colitis in adult mice. These data provide strong evidence that primary afferent neurons of the murine colon are a hitherto unexpected source of NGF in the normal and inflamed bowel. This work was supported by the Canadian Institutes of Health Research.

Homage to Rita Levi-Montalcini, the queen of modern neuroscience

The first cell growth factor, nerve growth factor (NGF), was discovered by Rita Levi-Montalcini (RLM) in the early 1950's in Washington University in Saint Louis, Missouri, USA. Originally identified as neurite outgrowth-stimulating factor, later studies revealed that non-neuronal cells, including immune cells, endothelial cells, cardiomyocytes, pancreatic beta cells, prostate epithelial cells and adipose tissue cells, are also targets for and/or sources of NGF. Nerve growth factor is well recognized at present to mediate multiple biological phenomena, ranging from the neurotrophic through immunotrophic and epitheliotrophic to metabotrophic effects. Consequently, NGF and other members of the neurotrophin family are implicated in the pathogenesis of a large spectrum of neuronal and non-neuronal diseases, ranging from Alzheimer's and other neurodegenerative diseases to atherosclerosis and other cardiometabolic diseases. Recent studies demonstrated the therapeutic potentials of NGF in these diseases including ocular and cutaneous diseases. Whereas NGF TrkA receptor antagonists emerged as novel drugs for pain, prostate and breast cancer, and urinary bladder syndromes. Here we briefly describe (i) the unpredictable ideogenesis of the discovery of NGF, and (ii) our scientific and human experience working in RML's laboratory for 15 years (GNC) and over 40 years (LA). Biomedical Reviews 2012; 23: 1-7.

NGF is involved in oral ovalbumin‐induced altered colonic contractility in rats: evidence from the blockade of TrkA receptors with K252a

Nerve growth factor (NGF)-mucosal mast cell (MMC) interaction has been implicated in the remodeling of enteric circuitries and associated functional changes. We investigated the involvement of NGF and its receptor TrkA in the altered colonic contractile activity observed in the model of oral ovalbumin (OVA)-induced MMC hyperactivity in rats. We also studied the role of colonic MMCs as a source of NGF. Rats received oral OVA, alone or with the TrkA antagonist K252a. Colonic co-expression of NGF/TrkA and rat mast cell protease II (RMCPII) (double immunofluorescence), RMCPII content (ELISA) and expression of NGF, Brain-derived neurotrophic factor (BDNF) and TrkA/B (QT-PCR) were assessed. Colonic contractile activity was determined in vivo and in vitro. TrkA, but not NGF, was localized in colonic MMCs (RMCPII-positive). Oral ovalbumin exposure increased colonic RMCPII levels but did not change the percentage of TrkA-positive MMCs. Neither OVA nor K252a, alone or combined, altered NGF, BDNF or TrkA/B expression. Spontaneous colonic activity in vivo and in vitro was altered by OVA, an effect prevented by K252a. Electrical stimulation-induced contractile responses in vivo and carbachol responses in vitro were increased by OVA in a K252a-independent manner. In OVA-treated animals, inhibition of NO synthesis with l-NNA significantly enhanced spontaneous colonic activity in vitro, a response completely prevented by K252a. These results suggest that NGF-TrkA-dependent pathways are implicated in colonic contractile alterations observed during OVA exposure in rats. NGF-TrkA system might represent a potential target for treatment of gastrointestinal disorders characterized by colonic motor alterations.

Neurotrophic Factor Changes in the Rat Thick Skin following Chronic Constriction Injury of the Sciatic Nerve

BackgroundCutaneous peripheral neuropathies have been associated with changes of the sensory fiber innervation in the dermis and epidermis. These changes are mediated in part by the increase in local expression of trophic factors. Increase in target tissue nerve growth factor has been implicated in the promotion of peptidergic afferent and sympathetic efferent sprouting following nerve injury. The primary source of nerve growth factor is cells found in the target tissue, namely the skin. Recent evidence regarding the release and extracellular maturation of nerve growth factor indicate that it is produced in its precursor form and matured in the extracellular space. It is our hypothesis that the precursor form of nerve growth factor should be detectable in those cell types producing it. To date, limitations in available immunohistochemical tools have restricted efforts in obtaining an accurate distribution of nerve growth factor in the skin of naïve animals and those with neuropathic pain lesions. It is the objective of this study to delineate the distribution of the precursor form of nerve growth factor to those cell types expressing it, as well as to describe its distribution with respect to those nerve fibers responsive to it.ResultsWe observed a decrease in peptidergic fiber innervation at 1 week after the application of a chronic constriction injury (CCI) to the sciatic nerve, followed by a recovery, correlating with TrkA protein levels. ProNGF expression in CCI animals was significantly higher than in sham-operated controls from 1-4 weeks post-CCI. ProNGF immunoreactivity was increased in mast cells at 1 week post-CCI and, at later time points, in keratinocytes. P75 expression within the dermis and epidermis was significantly higher in CCI-operated animals than in controls and these changes were localized to neuronal and non-neuronal cell populations using specific markers for each.ConclusionsWe describe proNGF expression by non-neuronal cells over time after nerve injury as well as the association of NGF-responsive fibers to proNGF-expressing target tissues. ProNGF expression increases following nerve injury in those cell types previously suggested to express it.

Molecular diversity of snake venom nerve growth factors

Nerve growth factor (NGF) is a protein which stimulates the differentiation and maintenance of sympathetic and embryonic sensory neurons. Snake venoms are a rich source of NGF. Due to small quantities it is sometimes difficult and laborious to isolate NGF from the venoms. In this study the use of Ni-NTA-agarose for isolation of NGF is studied. Anti- Vipera lebetina NGF antibodies were used for identification of NGF during Ni-NTA-agarose fractionation as well as for cross-reaction studies with 21 snake venoms. All studied venoms contained NGF. The molecular masses of the NGFs from Echis ocellatus, Agkistrodon contortrix contortrix, A. bilineatus, A. blomhoffii, A. saxatilis, Calloselasma rhodostoma, Bothrops jararaca and B. lanceolatus were determined for the first time. Some previous results of the NGF studies are revaluated.

Natural antibodies against nerve growth factor inhibit in vitro prostate cancer cell metastasis.

Prostate cancer is a major cause of death in older men, and bone metastasis is the primary cause of morbidity and mortality in prostate cancer. Prostate is an abundant source of nerve growth factor (NGF) that is secreted by malignant epithelial cells and utilized as an important autocrine factor for growth and metastasis. We previously showed that intravenous gammaglobulin (IVIg) contains natural antibodies against NGF, which inhibit growth and differentiation of the NGF-dependent cell line PC-12. In the present study, we examined the effects of these natural antibodies on in vitro migration or metastasis of two prostate cancer cell lines namely DU-145 and PC-3. Cancer cell migration was assessed using these cell lines in the upper chambers of Matrigel invasion chambers. The effects of IVIg and affinity-purified anti-NGF antibodies on cell migration through membrane into the lower chamber were assessed in dose/response experiments by a colorimetric method. Affinity-purified natural IgG anti-NGF antibody inhibited DU-145 migration by 38% (p=0.01) and PC-3 migration by 25% (p=0.02); whereas, a monoclonal anti-NGF antibody inhibited DU-145 migration by 40% (p=0.01) and PC-3 migration by 37% (p=0.02), at the same concentration. When IVIg was depleted of NGF-specific IgG by affinity chromatography, there was no significant inhibition of migration of the DU-145 and PC-3 cells at a concentration of 1mg/well. Removal of the NGF-specific antibody from the IVIg was also demonstrated by a lack of effect on PC-12 cell differentiation. Therefore, IVIg is able to inhibit the migration of prostate cancer cell lines, through Matrigel chambers in vitro, only when the natural NGF-specific antibodies actively are present in IVIg.

475j Nerve Growth and Plasticity in the Colonic Mucosa of Patients With Irritable Bowel Syndrome

Background & Aims: Immune and non-immune mediators released by gut mucosa play a role in the pathophysiology of irritable bowel syndrome (IBS). In this context, we hypothesize that soluble mediators induce phenotypic changes in gut mucosal innervation in patients with IBS. The aims of the present study were to evaluate in the colonic mucosa of IBS patients compared with healthy controls (HC): 1) Phenotypic changes in the colonic innervation, including the expression of the marker of neuronal sprouting growth-associated protein 43 (GAP-43); 2) source and expression of nerve growth factor (NGF); 3) impact of colonic mediators and NGF on neuronal cell differentiation and sprouting in human cultured neuronal cells. Methods: Mucosal biopsies were obtained from the descending colon of 18 IBS patients and 8 HC. The expression of neuronal specific enolase (NSE), GAP-43 and NGF expression were assessed by quantitative immunohistochemistry. Mucosal mediator release was quantified on colonic biopsies by means of specific immuno-enzymatic assay and/or semi-quantitative western blot. The impact ofmucosal supernatants on nerve sprouting and differentiation was assessed on cultured neuronal cell lines (SH-Sy5Y) by means of morphometric analysis. Results: IBS patients showed a significant increase in the area of lamina propria occupied by NSE+ fibers as compared to HC (2.52% vs 1.48%; P<0.05). A significant increase in GAP-43+ fibers was documented in IBS patients in comparison with HC (1.75% vs 1.05%; P<0.05). NGF immunoreactivity was significantly increased in IBS patients (1.31% vs 0.79%; P<0.05). The majority of NGF+ cells also expressed tryptase immunolabeling, suggesting that mucosal mast cells represent the main source of NGF. NGF mucosal concentration and protein expression was significantly increased in most IBS samples in comparison with HC. Mucosal supernatants obtained from IBS patients induced a 60% neuronal sprouting on cultured neuronal cell lines, which was similar to that obtained with the control stimulus retinoic acid (68%) and significantly higher than that obtained with HC (41%) (P<0.05). Neuronal sprouting was partly NGF-dependent, as it was significantly reduced by NGF neutralization. Conclusions: Nerve fiber density and growth are increased in IBS patients. NGF expression, mainly from mast cell source, is also increased. Mast cellderived NGF participates to neuronal sprouting. Our results provide a new mechanism which may contribute to the pathophysiology of IBS and identify new targets for novel therapies in patients with IBS.

475k A New Gastric-Emptying Mouse Model Based on In Vivo Non-Invasive Bioluminescence Functional Imaging

Background & Aims: Immune and non-immune mediators released by gut mucosa play a role in the pathophysiology of irritable bowel syndrome (IBS). In this context, we hypothesize that soluble mediators induce phenotypic changes in gut mucosal innervation in patients with IBS. The aims of the present study were to evaluate in the colonic mucosa of IBS patients compared with healthy controls (HC): 1) Phenotypic changes in the colonic innervation, including the expression of the marker of neuronal sprouting growth-associated protein 43 (GAP-43); 2) source and expression of nerve growth factor (NGF); 3) impact of colonic mediators and NGF on neuronal cell differentiation and sprouting in human cultured neuronal cells. Methods: Mucosal biopsies were obtained from the descending colon of 18 IBS patients and 8 HC. The expression of neuronal specific enolase (NSE), GAP-43 and NGF expression were assessed by quantitative immunohistochemistry. Mucosal mediator release was quantified on colonic biopsies by means of specific immuno-enzymatic assay and/or semi-quantitative western blot. The impact ofmucosal supernatants on nerve sprouting and differentiation was assessed on cultured neuronal cell lines (SH-Sy5Y) by means of morphometric analysis. Results: IBS patients showed a significant increase in the area of lamina propria occupied by NSE+ fibers as compared to HC (2.52% vs 1.48%; P<0.05). A significant increase in GAP-43+ fibers was documented in IBS patients in comparison with HC (1.75% vs 1.05%; P<0.05). NGF immunoreactivity was significantly increased in IBS patients (1.31% vs 0.79%; P<0.05). The majority of NGF+ cells also expressed tryptase immunolabeling, suggesting that mucosal mast cells represent the main source of NGF. NGF mucosal concentration and protein expression was significantly increased in most IBS samples in comparison with HC. Mucosal supernatants obtained from IBS patients induced a 60% neuronal sprouting on cultured neuronal cell lines, which was similar to that obtained with the control stimulus retinoic acid (68%) and significantly higher than that obtained with HC (41%) (P<0.05). Neuronal sprouting was partly NGF-dependent, as it was significantly reduced by NGF neutralization. Conclusions: Nerve fiber density and growth are increased in IBS patients. NGF expression, mainly from mast cell source, is also increased. Mast cellderived NGF participates to neuronal sprouting. Our results provide a new mechanism which may contribute to the pathophysiology of IBS and identify new targets for novel therapies in patients with IBS.