High-affinity Nerve Growth Factor Receptor Research Articles

Immunohistochemical phenotype of sensory neurons associated with sympathetic plexuses in the trigeminal ganglia of adult nerve growth factor transgenic mice.

Following peripheral nerve injury, postganglionic sympathetic axons sprout into the affected sensory ganglia and form perineuronal sympathetic plexuses with somata of sensory neurons. This sympathosensory coupling contributes to the onset and persistence of injury-induced chronic pain. We have documented the presence of similar sympathetic plexuses in the trigeminal ganglia of adult mice that ectopically overexpress nerve growth factor (NGF), in the absence of nerve injury. In this study, we sought to further define the phenotype(s) of these trigeminal sensory neurons having sympathetic plexuses in our transgenic mice. Using quantitative immunofluorescence staining analyses, we show that the invading sympathetic axons specifically target sensory somata immunopositive for several biomarkers: NGF high-affinity receptor tyrosine kinase A (trkA), calcitonin gene-related peptide (CGRP), neurofilament heavy chain (NFH), and P2X purinoceptor 3 (P2X3). Based on these phenotypic characteristics, the majority of the sensory somata surrounded by sympathetic plexuses are likely to be NGF-responsive nociceptors (i.e., trkA expressing) that are peptidergic (i.e., CGRP expressing), myelinated (i.e., NFH expressing), and ATP sensitive (i.e., P2X3 expressing). Our data also show that very few sympathetic plexuses surround sensory somata expressing other nociceptive (pain) biomarkers, including substance P and acid-sensing ion channel 3. No sympathetic plexuses are associated with sensory somata that display isolectin B4 binding. Though the cellular mechanisms that trigger the formation of sympathetic plexus (with and without nerve injury) remain unknown, our new observations yield an unexpected specificity with which invading sympathetic axons appear to target a precise subtype of nociceptors. This selectivity likely contributes to pain development and maintenance associated with sympathosensory coupling.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Piezo2 Knockdown Inhibits Noxious Mechanical Stimulation and NGF-Induced Sensitization in A-Delta Bone Afferent Neurons.

Piezo2 is a mechanically gated ion-channel that has a well-defined role in innocuous mechanical sensitivity, but recently has also been suggested to play a role in mechanically induced pain. Here we have explored a role for Piezo2 in mechanically evoked bone nociception in Sprague Dawley rats. We have used an in vivo electrophysiological bone-nerve preparation to record the activity of single Aδ bone afferent neurons in response to noxious mechanical stimulation, after Piezo2 knockdown in the dorsal root ganglia with intrathecal injections of Piezo2 antisense oligodeoxynucleotides, or in control animals that received mismatch oligodeoxynucleotides. There were no differences in the number of Aδ bone afferent neurons responding to the mechanical stimulus, or their threshold for mechanical activation, in Piezo2 knockdown animals compared to mismatch control animals. However, bone afferent neurons in Piezo2 knockdown animals had reduced discharge frequencies and took longer to recover from stimulus-evoked fatigue than those in mismatch control animals. Piezo2 knockdown also prevented nerve growth factor (NGF)-induced sensitization of bone afferent neurons, and retrograde labeled bone afferent neurons that expressed Piezo2 co-expressed TrkA, the high affinity receptor for NGF. Our findings demonstrate that Piezo2 contributes to the response of bone afferent neurons to noxious mechanical stimulation, and plays a role in processes that sensitize them to mechanical stimulation.

Interleukin-10 Reduces Neurogenic Inflammation and Pain Behavior in a Mouse Model of Type 2 Diabetes.

PurposeNeurogenic inflammation is a major component of chronic neuropathic pain. Previously, we established the db/db mouse as an animal model of painful diabetic neuropathy (PDN) of type 2 diabetes. In the current study, we investigate the roles of interleukin (IL)-10, an anti-inflammatory cytokine, in the development of neurogenic inflammation and pain behavior in db/db mouse.Materials and MethodsWe first studied IL-10 expression in lumbar dorsal root ganglion (LDRG) neurons of db/db mice using immunohistochemistry, immunoblots, and reverse transcription polymerase chain reaction during the period of pain behavior (from 8 to 16 wk of age). To determine if the reduced IL-10 expression mediates the mechanical allodynia in db/db mice, we administered recombinant mouse IL-10 or saline (control) intraperitoneally to control db/+ and db/db mice starting at 8 wk of age. IL-10 treatment was repeated every other day for 2 wk until the mice reached 10 wk of age.ResultsDuring the period of pain behavior, reduction of IL-10 protein and gene expression was detected in LDRG of db/db mice. Treatment with recombinant IL-10, from 8 to 10 wk of age, alleviates pain behaviors in db/db mice without affecting other diabetic parameters. In parallel, IL-10 treatment reduced the upregulation of nerve growth factor (NGF), inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and high-affinity NGF receptor (Trk A) in LDRG, as well as the numbers of iNOS-positive Langerhans cells and CD-68-positive dermal dendritic cells in the hind-foot-pad skin in db/db mice.ConclusionOur findings suggest that the reduction in neuronal IL-10 increases inflammatory phenomena, ultimately contributing to PDN. These results suggest that the dysregulation of cytokine-mediated inflammation contributes to the development of PDN in db/db mice. Targeting this pathophysiologic mechanism could be an effective approach for treating PDN in type 2 diabetes.

Neuroprotection by Neurotropin through Crosstalk of Neurotrophic and Innate Immune Receptors in PC12 Cells.

Infected or damaged tissues release multiple “alert” molecules such as alarmins and damage-associated molecular patterns (DAMPs) that are recognized by innate immune receptors, and induce tissue inflammation, regeneration, and repair. Recently, an extract from inflamed rabbit skin inoculated with vaccinia virus (Neurotropin®, NTP) was found to induce infarct tolerance in mice receiving permanent ischemic attack to the middle cerebral artery. Likewise, we report herein that NTP prevented the neurite retraction in PC12 cells by nerve growth factor (NGF) deprivation. This effect was accompanied by interaction of Fyn with high-affinity NGF receptor TrkA. Sucrose density gradient subcellular fractionation of NTP-treated cells showed heretofore unidentified membrane fractions with a high-buoyant density containing Trk, B subunit of cholera toxin-bound ganglioside, flotillin-1 and Fyn. Additionally, these new membrane fractions also contained Toll-like receptor 4 (TLR4). Inhibition of TLR4 function by TAK-242 prevented the formation of these unidentified membrane fractions and suppressed neuroprotection by NTP. These observations indicate that NTP controls TrkA-mediated signaling through the formation of clusters of new membrane microdomains, thus providing a platform for crosstalk between neurotrophic and innate immune receptors. Neuroprotective mechanisms through the interaction with innate immune systems may provide novel mechanism for neuroprotection.

Brainstem expression of SLC6A4, HTR2C, NGF, BDNF, TRKANGF, TRKBBDNF and P75NTR following paternal alcohol exposure in the male mouse

We previously showed in the mouse that paternal preconception alcohol exposure (PPAE) affects alcohol sensitivity by analyzing postnatal alcohol preference in the offspring. In this mouse study by using the same animals of the previous investigation we aimed at examining whether or not PPAE may disrupt the epigenetic regulation of postnatal alcohol sensitivity in the offspring by investigating pathways regulating mood, emotion, serotonergic tone and neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). We analyzed the brainstem gene expression of serotonin transporter Solute Carrier Family 6 Member4 (SLC6A4), 5-Hydroxytryptamine Receptor 2C (HTR2C) binding the neurotransmitter serotonin, and NGF, BDNF and their tropomyosin receptor kinase A (TrkANGF) and B (TrKBBDNF) (high-affinity NGF and BDNF receptors) and p75NTR (low-affinity, pan-neurotrophins receptor) in adult offspring that underwent or not postnatal alcohol exposure. We found SLC6A4 elevation and decreased HTR2C in the offspring of chronic alcohol-exposed sires. We also disclosed p75NTR elevation in the offspring of chronically exposed sires as well as postnatal sensitization to low alcohol doses in the offspring of chronically exposed sires for both TrKBBDNF and BDNF. In our PPAE mouse model, where genotype effects can be carefully measured, we observed that the sires’ exposure to alcohol before mating might disrupt the sensitivity to the serotonergic/neurotrophic-associated effects of alcohol influencing the postnatal alcohol preference in the offspring.

Evidence for the Presence of Long-Lived Plasma Cells in Nasal Polyps.

PurposePlasma cells and immunoglobulins (Igs) play a pivotal role in the induction and maintenance of chronic inflammation in nasal polyps. During secondary immune responses, plasma cell survival and Ig production are regulated by the local environment. The purpose of the present study was to investigate the presence of long-lived plasma cells (LLPCs) and specific survival niches for LLPCs in human nasal polyps.MethodsNasal mucosal samples were cultured with an air-liquid interface system and the Ig levels in culture supernatants were analyzed by enzyme-linked immunosorbent assay. The characteristics of LLPCs in nasal polyps were determined by immunohistochemistry and immunofluorescence. The expression of neurotrophins as well as their receptors was detected by quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and Western blotting.ResultsThe numbers of CD138+ total plasma cells and BCL2+ plasma cells were increased in both eosinophilic and non-eosinophilic nasal polyps compared with those in normal tissues. The production of IgG, IgA, and IgE was detected in culture supernatants even after a 32-day culture of nasal polyps. Although the total numbers of plasma cells were decreased in nasal polyps after culture, the numbers of BCL2+ plasma cells remained stable. The expression of nerve growth factor (NGF) as well as tropomyosin receptor kinase (Trk) A, a high-affinity receptor for NGF, was upregulated in both eosinophilic and non-eosinophilic nasal polyps. In addition, BCL2+ plasma cell numbers were positively correlated with NGF and TrkA mRNA expression in nasal mucosal tissues. Polyp plasma cells had the expression of TrkA.ConclusionsHuman nasal polyps harbor a population of LLPCs and NGF may be involved in their prolonged survival. LLPCs may be a novel therapeutic target for suppressing the local Ig production in nasal polyps.

Tumor-derived exosomes educate fibroblasts to promote salivary adenoid cystic carcinoma metastasis via NGF-NTRK1 pathway.

Salivary adenoid cystic carcinoma (SACC) is a rare head and neck malignancy characterized by unpredictable expansion, considerable perineural invasion and high risk of metastasis; however, the underlying mechanism of SACC progression remains unclear. Cancer-associated fibroblasts localized within the tumor microenvironment may promote cancer malignant transformation by enhancing tumor growth, blood vessel formation, inflammation development and metastasis occurrence. Small extracellular vesicles, including exosomes, are mediators of intercellular communication and can influence major tumor-associated pathways. The present study aimed to explore the exosome-mediated communication between SACC cells and fibroblasts. The results from confocal microscopy demonstrated that exosomes derived from the human cell line SACC-83 were internalized by the human periodontal ligament fibroblast (HPLF) cells. Following exosome internalization, HPLF cells appeared to enhance SACC-83 cell metastasis and were educated toward a protumorigenesis phenotype according to transcriptome RNA sequencing and reverse transcription-quantitative polymerase chain reaction analysis. This phenomenon included exosome-mediated stimulation of proinflammatory cytokines and nerve growth factor (NGF) secretion. Furthermore, NGF blockage reduced the enhanced SACC-83 cell invasion stimulated by the supernatant isolated from exosome-educated HPLF cells. In addition, the results reported that neurotrophic receptor tyrosine kinase 1 (NTRK1), which is the high-affinity NGF receptor, was significantly upregulated in human SACC-83 cells. These results demonstrated that SACC-83 cell-derived exosomes educated HPLF cells toward the protumorigenic phenotype via the NGF-NTRK1 pathway, which suggested that this type of exosomes may be used as a potential therapeutic target for SACC.

Role of NGF-TrkA signaling in calcification of articular chondrocytes.

Nerve growth factor (NGF) is a key regulator of chronic osteoarthritic pain, but the exact targets of NGF action on human articular cartilage is unknown. This study aimed to test the hypothesis that the NGF-tropomyosin receptor kinase A (TrkA) (high-affinity NGF receptor) pathway plays a role in the calcification process of human articular chondrocytes (hACs). A 14-aa small peptide of NGF (Nsp) previously shown to activate NGF signaling in rat PC12 cells was used as an NGF signaling agonist, and recombinant NGF and the pan-Trk inhibitor GNF-5837 were employed as signaling modulating agents. The functional consequences of NGF-TrkA signaling were examined in human healthy articular chondrocytes maintained under conditions supportive of osteogenesis in vitro. The NGF-mimetic bioactivity of Nsp was first confirmed on the basis of maintenance of neurite outgrowth in PC12 cells. Primary human chondrocytes responded to Nsp in vitro. Perturbation of NGF signaling with NGF, Nsp, and GNF-5837 resulted in a strong induction of chondrocyte calcification, and gene expression data suggested that the Indian Hedgehog-parathyroid hormone-related protein signaling axis was involved. These findings suggest functional involvement of NGF signaling in calcification of hACs and the importance of NGF signaling in articular cartilage homeostasis.-Jiang, Y., Tuan, R. S. Role of NGF-TrkA signaling in calcification of articular chondrocytes.

Aberrant Expression of Histamine-independent Pruritogenic Mediators in Keratinocytes may be Involved in the Pathogenesis of Prurigo Nodularis.

Prurigo nodularis is a highly pruritic and hyperplastic chronic dermatosis with unknown pathogenesis. Many pruritogenic mediators, including nerve growth factor, interleukin (IL)-31, thymic stromal lymphopoietin, and endothelin-1, are implicated in chronic itch and inflammation. This study investigated the mRNA levels and immunoreactivity of the nerve growth factor, IL-31, thymic stromal lymphopoietin, and endothelin axes in both lesional and perilesional skin in prurigo nodularis by using quantitative real-time PCR and immunohistochemistry studies. The nerve growth factor high-affinity receptor tyrosine kinase receptor A was upregulated while the low affinity receptor p75 neurotrophin receptor was downregulated in prurigo nodularis lesions. Downregulated expression of IL-31/IL-31 receptor A and endothelin-3/endothelin receptor B and upregulation of thymic stromal lymphopoietin receptor were found in prurigo nodularis lesions. Aberrant expression of nerve growth factor, IL-31, thymic stromal lymphopoietin and endothelin axes was found in prurigo nodularis lesions, especially in the epidermis, indicating the importance of keratinocytes in prurigo nodularis pathogenesis.

Transdifferentiation effects and related mechanisms of nerve growth factor and internal limiting membrane on Müller cells

In this study, we investigated the pro-proliferative, pro-transdifferentiation effects and mechanisms of nerve growth factor (NGF), internal limiting membrane (ILM), and NGF+ILM on Müller cells. The Müller cells cultured with NGF, ILM or both were mediated with tyrosine kinase A (TrkA) (a high affinity receptor for NGF) inhibitor, phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) (an intracellular signaling pathway important in regulating the cell cycle) inhibitor, or LIN28 (a RNA-binding protein and a posttranscriptional regulator of genes involved in developmental timing and self-renewal in embryonic stem cells) siRNA. Immunofluorescence (IF), western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect the expression of related genes. As a result, NGF, ILM and NGF+ILM promoted cell proliferation, increased the ratio of 5-bromo-2-deoxyuridine (BrdU)-positive cells, and were correlated with TrkA and PI3K/Akt signaling. NGF alone promoted cell dedifferentiation and redifferentiation mainly towards neurons rather than towards glial cells, related to TrkA and PI3K/Akt signals. The expression of p-Akt and cyclinD1 was increased by the intervention of NGF, ILM or NGF+ILM via TrkA and PI3K/Akt signals. NGF alone promoted the expression of paired box 6 (PAX6) (a transcription factor present during embryonic development), sex-determining region Y-box 2 (SOX2) (a transcription factor essential for the self-renewal, or pluripotency, of undifferentiated embryonic stem cells) and LIN28, and inhibited the expression of lethal-7 (Let-7b), Let-7d, Let-7i and miR-98 (microRNAs, key developmental regulators). The expression of Achaete-scute homolog 1 (Ascl1, also called MASH1) (a neurodevelopmental gene) and endogenous NGF (closely related to neurogenesis) was also promoted by exogenous NGF and related to TrkA and PI3K/Akt signaling. The down-regulation of LIN28 significantly antagonized the effects of NGF on the transdifferentiation of Müller cells. Over all, our results showed that NGF promoted the proliferation and transdifferentiation of Müller cells towards photoreceptor neurons--not towards glial cells, which was related to the LIN28/Let-7 pathway through TrkA and PI3K/Akt signals. Additionally, ILM promoted Müller cells to enter the cell cycle and enhanced cell proliferation, since NGF+ILM promoted the proliferation of Müller cell more significantly than NGF alone.

Adolescent binge ethanol-induced loss of basal forebrain cholinergic neurons and neuroimmune activation are prevented by exercise and indomethacin.

Basal forebrain cholinergic neurons mature in adolescence coinciding with development of adult cognitive function. Preclinical studies using the rodent model of adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-days on/2-days off from postnatal day [P]25 to P55) reveal persistent increases of brain neuroimmune genes that are associated with cognitive dysfunction. Adolescent intermittent ethanol exposure also reduces basal forebrain expression of choline acetyltransferase (ChAT), an enzyme critical for acetylcholine synthesis in cholinergic neurons similar to findings in the post-mortem human alcoholic basal forebrain. We report here that AIE decreases basal forebrain ChAT+IR neurons in both adult female and male Wistar rats following early or late adolescent ethanol exposure. In addition, we find reductions in ChAT+IR somal size as well as the expression of the high-affinity nerve growth factor (NGF) receptor tropomyosin receptor kinase A (TrkA) and the low-affinity NGF receptor p75NTR, both of which are expressed on cholinergic neurons. The decrease in cholinergic neuron marker expression was accompanied by increased phosphorylation of NF-κB p65 (pNF-κB p65) consistent with increased neuroimmune signaling. Voluntary wheel running from P24 to P80 prevented AIE-induced cholinergic neuron shrinkage and loss of cholinergic neuron markers (i.e., ChAT, TrkA, and p75NTR) as well as the increase of pNF-κB p65 in the adult basal forebrain. Administration of the anti-inflammatory drug indomethacin (4.0 mg/kg, i.p prior to each ethanol exposure) during AIE also prevented the loss of basal forebrain cholinergic markers and the concomitant increase of pNF-κB p65. In contrast, treatment with the proinflammatory immune activator lipopolysaccharide (1.0 mg/kg, i.p. on P70) caused a loss of cholinergic neuron markers that was paralleled by increased pNF-κB p65 in the basal forebrain. These novel findings are consistent with AIE causing lasting activation of the neuroimmune system that contributes to the persistent loss of basal forebrain cholinergic neurons in adulthood.

Human carbonic anhydrase-8 AAV8 gene therapy inhibits nerve growth factor signaling producing prolonged analgesia and anti-hyperalgesia in mice.

Carbonic anhydrase-8 (Car8; murine gene symbol) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates neuronal intracellular calcium release. We previously reported that wildtype Car8 overexpression corrects the baseline allodynia and hyperalgesia associated with calcium dysregulation in the waddle (wdl) mouse due to a 19 bp deletion in exon 8 of the Car8 gene. In this report, we provide preliminary evidence that overexpression of the human wildtype ortholog of Car8 (CA8WT), but not the reported CA8 S100P loss-of-function mutation (CA8MT); inhibits nerve growth factor (NGF)-induced phosphorylation of ITPR1, TrkA (NGF high affinity receptor); and ITPR1-mediated cytoplasmic free calcium release in vitro. Additionally, we show that gene-transfer using AAV8-V5-CA8WT viral particles via sciatic nerve injection demonstrates retrograde transport to dorsal root ganglia (DRG) producing prolonged V5-CA8WT expression, pITPR1 and pTrkA inhibition, and profound analgesia and anti-hyperalgesia in male C57BL/6J mice. AAV8-V5-CA8WT mediated overexpression prevented and treated allodynia and hyperalgesia associated with chronic neuropathic pain produced by the spinal nerve ligation (SNL) model. These AAV8-V5-CA8 data provide a proof-of-concept for precision medicine through targeted gene therapy of NGF-responsive somatosensory neurons as a long-acting local analgesic able to prevent and treat chronic neuropathic pain through regulating TrkA signaling, ITPR1 activation, and intracellular free calcium release by ITPR1.

Asparagus cochinchinensis stimulates release of nerve growth factor and abrogates oxidative stress in the Tg2576 model for Alzheimer\u2019s disease

BackgroudUse of multifunctional drugs with neurotrophic supporting and oxidative stress suppressing activity may be considered a therapeutic strategy to protect or repair cellular damage caused during the progression of Alzheimer’s disease (AD). In this study, we investigated the therapeutic effects of aqueous extract of A. cochinchinesis root (AEAC), particularly its role as a nerve growth factor (NGF) stimulator and anti-oxidant in Tg2576 mice showing AD phenotypes of human.MethodsTg2576 mice were received 100 mg/kg/day AEAC via oral administration, while mice in the Vehicle treated group received dH2O for 4 weeks. Non-Tg littermates were used as a control group. Following AEAC treatment for 4 weeks, NGF function, anti-oxidantive status, Aβ-42 peptide level, γ-secretase expression and neuronal cell functions were analyzed in the brain of Tg2576 mice.ResultsAEAC containing flavonoids, phenols, saponins and protodioscin induced enhancement of NGF secretion and decreased intracellular ROS in the neuronal and microglial cell line. These effects as well as enhanced SOD levels were also detected in AEAC treated Tg2576 mice. The expression of p-Akt among downstream effectors of the high affinity NGF receptor was dramatically recovered in AEAC treated Tg2576 mice, while the expression of p75NTR was slightly recovered in the same group. Significant recovery on the level of Aβ-42 peptides and the expression of γ-secretase members including PS-2, APH-1 and NCT were detected in AEAC treated Tg2576 mice. Furthermore, AEAC treated Tg2576 mice showed decreased numbers of dead cells and suppressed acetyl choline esterase (AChE) activity.ConclusionsThese results suggest that AEAC contribute to improving the deposition of Aβ-42 peptides and neuronal cell injuries during the pathological progression stage of AD in the brain of Tg2576 mice through increased NGF secretion and suppressed oxidative stress.

Excessive nerve growth factor impairs bidirectional communication between the oocyte and cumulus cells resulting in reduced oocyte competence

BackgroundExcessive nerve growth factor (NGF) is commonly found in the follicular fluid of patients with polycystic ovary syndrome (PCOS). Furthermore, oocytes from PCOS patients exhibit lower developmental competence. The purpose of this study was to explore the association between excessive NGF and low oocyte competence in vitro.MethodsExcessive NGF was added to mouse cumulus oocyte complexes (COCs) cultured in vitro to investigate meiotic maturation of the oocyte. After culture, mRNA expression levels of Pfkp and Ldha genes in cumulus cells (CCs) and Gdf9, Bmp15 and Fgf8 genes in oocytes, were determined by real-time quantitative polymerase chain reaction (qPCR). We also investigated the mRNA content of Pfkp and Ldha in CCs from PCOS and non-PCOS patients.ResultsExcessive NGF significantly inhibited oocyte meiotic maturation. The inhibitory effect was mediated by the NGF high-affinity receptor, NTRK1. mRNA content of Pfkp and Ldha genes in CCs was significantly reduced by excessive NGF stimulation. Moreover, the expression levels of Gdf9, Bmp15 and Fgf8 were also decreased in oocytes, and was induced by excessive NGF-stimulated CCs. In addition, lower expression levels of Pfkp and Ldha in CCs were identified in Chinese PCOS patients with excessive NGF (PCOS, 22 ± 2.63 ng/ml, n = 13; non-PCOS, 7.18 ± 2.42 ng/ml, n = 9; p < 0.01) in the follicular fluid, suggesting a potential association between excessive NGF and decreased glycolysis in the CCs of women with PCOS.ConclusionsExcessive NGF impairs bidirectional communication between oocyte and cumulus cells, which might be related to low oocyte competence.

Effect of nerve growth factor on osteogenic potential of type 2 diabetic mice bone marrow stromal cell in vitro

Objective: To study the effects of nerve growth factor (NGF) on the proliferation, osteogenic differentiation and mineralization of type 2 diabetic mice bone marrow stromal cell (BMSC), providing basis for clinical application of NGF. Methods: Three 8-week-old male db/db mice and two 8-week-old male C57BL/6J mice were used in the study. BMSC derived from femur were cultured though adherence method. BMSC of C57BL/6J mice and db/db mice was divided into normal group and diabetic group to conduct the osteogenic potential experiment, named experiment one. In experiment two, diabetic BMSC was divided into 3 groups: diabetic control group, NGF group, and K252a+NGF group [K252a was the inhibitor of tyrosine kinase A (TrkA), which was the high affinity receptor of NGF], to investigate effect of NGF on osteogenic potential of diabetic mice BMSC. After seeding BMSC, K252a was added into K252a+NGF group, then NGF was added 30 min later. NGF was added into NGF group and K252a+NGF group, but not diabetic control group. The proliferation of BMSC at 1, 3, 5 and 7 d in experiment one and the proliferation of BMSC at 1, 2 and 3 d in experiment two were evaluated through methyl thiazolyl tetrazolium, and the level of alkaline phosphatase (ALP) at 3, 5 and 7 d in both experiments were measured. After being osteogenic induced for 14 d, mineralized nodules in both experiments were quantitated by alizarin red calcium stain. Five holes were set in every group, and all experiments were repeated 3 times. Results: The BMSC proliferation of diabetic group was significantly higher than that of the normal group at 3, 5 and 7 d (P<0.05). After being osteogenic inducted for 3, 5 and 7 d, ALP level of diabetic group were significantly lower than that of normal group (P<0.05). After being osteogenic inducted for 14 d, calcium nodule count of diabetic group [(23.1±6.4) nodule/field] were significantly lower than that of normal group [(36.9±7.9) nodule/field](P<0.05). At 1, 2 and 3 d, BMSC proliferations of diabetic control group, NGF group and K252a+NGF group were not statistically different (P>0.05). After being osteogenic inducted for 3 and 5 d, ALP level of NGF group was significantly higher than that of diabetic control group (P<0.05). After being osteogenic inducted for 3, 5, and 7 d, ALP level of K252a+NGF group was significantly lower than that of NGF group (P<0.05) and diabetic control group (P<0.05). After being osteogenic induced for 14 d, calcium nodule count of NGF group [(45.2±6.8) nodule/field] was significantly more than that of diabetic control group [(23.1±6.4) nodule/field](P<0.05); while calcium nodule count of K252a+NGF group [(18.0±4.5) nodule/field] was significantly less than that of NGF group (P<0.05) and diabetic control group (P<0.05). Conclusions: The differentiation and mineralization of type 2 diabetic mice BMSC was significantly reduced. NGF promoted the osteoblastic differentiation and mineralization of diabetic mice BMSC in viro though combining with TrkA.

Young Human Cholinergic Neurons Respond to Physiological Regulators and Improve Cognitive Symptoms in an Animal Model of Alzheimer's Disease.

The degeneration of cholinergic neurons of the nucleus basalis of Meynert (NBM) in the basal forebrain (BF) is associated to the cognitive decline of Alzheimer’s disease (AD) patients. To date no resolutive therapies exist. Cell-based replacement therapy is a strategy currently under consideration, although the mechanisms underlying the generation of stem cell-derived NBM cholinergic neurons able of functional integration remain to be clarified. Since fetal brain is an optimal source of neuronal cells committed towards a specific phenotype, this study is aimed at isolating cholinergic neurons from the human fetal NBM (hfNBMs) in order to study their phenotypic, maturational and functional properties. Extensive characterization confirmed the cholinergic identity of hfNBMs, including positivity for specific markers (such as choline acetyltransferase) and acetylcholine (Ach) release. Electrophysiological measurements provided the functional validation of hfNBM cells, which exhibited the activation of peculiar sodium (INa) and potassium (IK) currents, as well as the presence of functional cholinergic receptors. Accordingly, hfNBMs express both nicotinic and muscarinic receptors, which were activated by Ach. The hfNBMs cholinergic phenotype was regulated by the nerve growth factor (NGF), through the activation of the high-affinity NGF receptor TrkA, as well as by 17-β-estradiol through a peculiar recruitment of its own receptors. When intravenously administered in NBM-lesioned rats, hfNBMs determined a significant improvement in memory functions. Histological examination of brain sections showed that hfNBMs (labeled with PKH26 fluorescent dye prior to administration) reached the damaged brain areas. The study provides a useful model to study the ontogenetic mechanisms regulating the development and maintenance of the human brain cholinergic system and to assess new lines of research, including disease modeling, drug discovery and cell-based therapy for AD.

NGF protects endothelial cells from indomethacin-induced injury through activation of mitochondria and upregulation of IGF-1

Background/aimsEndothelial cells (ECs) lining blood vessels are critical for delivery of oxygen and nutrients to all tissues and organs and play a crucial role in the regeneration of blood vessel following tissue injury. ECs are also major targets of injury by a variety of noxious factors [e.g., ethanol and nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin, diclofenac], especially in gastric mucosa that has direct exposure to these agents. In this study, we investigated whether nerve growth factor (NGF) can protect gastric microvascular ECs (GECs) from injury by indomethacin (INDO) and the mechanisms involved. MethodsGECs were isolated from rat gastric mucosa and pre-treated with either vehicle or NGF (100ng/ml) for 30min to 4h followed by treatment with vehicle or 0.25mM INDO for 4h. Studies1) cell viability using Calcein AM live cell tracking dye, 2) mitochondrial structure and function using MitoTracker, molecular probe that stains mitochondria in live cells in a manner dependent on mitochondrial membrane potential (MMP), 3) in vitro angiogenesis – endothelial tube formation on Matrigel, 4) expression and subcellular localization of NGF receptor, TrkA, and 5) expression of IGF-1 protein. ResultsTreatment with INDO reduced GEC viability and in vitro angiogenesis and induced mitochondrial injury and MMP depolarization. NGF pre-treatment protected GECs from INDO-induced injury preventing both INDO-induced MMP depolarization and reduced in vitro angiogenesis. The NGF high affinity receptor, TrkA, was localized in GECs to both cell membrane and mitochondria. NGF treatment of GECs also resulted in increased IGF-1 protein expression. Conclusions1) NGF protects GECs against IND-induced injury. 2) Mitochondria are major targets of both INDO-induced injury and NGF afforded protection of GECs. 3) TrkA expression in the mitochondria of GECs indicates that the protection afforded by NGF is partly mediated by its direct action on mitochondria. 4) NGF prevents MMP depolarization and increases expression of IGF-1 protein in GECs. These studies indicate that NGF may play a protective role against injury to GECs; and, that maintenance of mitochondrial structure and function is one of the mechanisms.

Morphological and immunohistochemical comparison of intrapancreatic nerves between chronic pancreatitis and type 1 autoimmune pancreatitis

ObjectivesThe abdominal pain associated with chronic pancreatitis (CP) may be related to the increased number and size of intrapancreatic nerves. On the other hand, patients with type 1 autoimmune pancreatitis (AIP) rarely suffer from the pain syndrome, and there are no previous studies concerning the histopathological findings of intrapancreatic nerves in patients with type 1 AIP. The current study is aimed at investigating the differences in the histopathological and immunohistochemical findings of intrapancreatic nerves in patients with CP and type 1 AIP. MethodsNeuroanatomical differences between CP and type 1 AIP were assessed by immunostaining with a pan-neuronal marker, protein gene product 9.5 (PGP9.5). The number (neural density) and area (neural hypertrophy) of PGP9.5-immunopositive nerves were quantitatively analyzed. Furthermore, the expression of nerve growth factor (NGF), and a high affinity receptor for NGF, tyrosine kinase receptor A (TrkA), was assessed by immunohistochemistry. ResultsBoth neural density and hypertrophy were significantly greater in pancreatic tissue samples from patients with CP than those with normal pancreas or type 1 AIP. NGF expression was stronger in type 1 AIP than in CP, whereas TrkA expression in type 1 AIP was poorer than in CP. ConclusionsAlthough CP and type 1 AIP are both characterized by the presence of sustained pancreatic inflammation, they are different in terms of the density and hypertrophy of intrapancreatic nerve fibers. It is possible that this may be related to the difference in the activity of the NGF/TrkA-pathway between the two types of pancreatitis.

신경세포에서 신경성장인자(nerve growth factor)의 조절에 미치는 천문동(Asparagus cochinchinensis) 열수추출물의 영향

Asparagus cochinchinensis is a medical plant that has long been used to treat fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease in northeast Asian countries. Although several studies have been conducted on the anti-neuroinflammatory effects of A. cochinchinensis, the correlation between these effects and nerve growth factor (NGF) has not yet been examined. In this study, we investigated the effects of an aqueous extract of A. cochinchinensis (AEAC) on the secretion and action mechanism of NGF in neuronal cells. The concentration of the NGF protein in the supernatant collected from cultured cells increased significantly in B35 cells treated with AEAC in comparison with the vehicle-treated group without any specific cytotoxicity. Furthermore, the mRNA expression of NGF showed a very similar pattern to its protein concentration. To examine the bioactivity of NGF secreted from B35 cells, undifferentiated PC12 cells were cultured in an AEAC-conditioned medium and neuritic outgrowth was observed. The dendrite length of PC12 cells in the AEAC-treated group was significantly higher than that in the vehicle-treated group. Moreover, the level of the downstream effectors p-TrkA and p-ERK of the high-affinity NGF receptor was significantly higher in the AEAC-treated group, while the expression of the downstream effectors of the low-affinity NGF receptor was significantly lower in the same group. These results suggest that AEAC may contribute to the regulation of NGF expression and secretion in neuronal cells; it is therefore an excellent candidate for further investigation as a therapeutic drug for neurodegenerative diseases.