Nerve Growth Factor Stimulation Research Articles

Hericium erinaceus, an amazing medicinal mushroom

Medicinal mushrooms have become a compelling topic because the bioactive compounds they contain promise a plethora of therapeutic properties. Hericium erinaceus commonly known as “Houtou” or “Shishigashira” in China and “Yamabushitake” in Japan, has commonly been prescribed in traditional Chinese medicine (TCM), because its consumption has been shown to be beneficial to human health. The species is found throughout the northern hemisphere in Europe, Asia, and North America. Hericium erinaceus has been firmly established as an important medicinal mushroom and its numerous bioactive compounds have been developed into food supplements and alternative medicines. However, the correspondence of the active components that cause the observed effects is often not clear. The mushroom as well as the fermented mycelia have been reported to produce several classes of bioactive molecules, including polysaccharides, proteins, lectins, phenols, and terpenoids. Most interestingly, two classes of terpenoid compounds, hericenones and erinacines, from fruiting bodies and cultured mycelia, respectively, have been found to stimulate nerve growth factor (NGF) synthesis. In this review we examine the scientific literature to explore and highlight the scientific facts concerning medicinal properties of H. erinaceus. We provide up-to-date information on this mushroom, including its taxonomy and a summary of bioactive compounds that appear related to the therapeutic potential of H. erinaceus.

Bioactive Diterpenoids from the Leaves of Callicarpa macrophylla.

A phytochemical investigation of the leaves of Callicarpa macrophylla led to the isolation of five new diterpenoids (1-5), macrophypenes A-E, and nine known analogues (6-14). The structures of 1-5 were established on the basis of extensive analysis of NMR spectroscopic data, X-ray diffraction data, and experimental and calculated electronic circular dichroism spectra. Compound 1 is a spiroditerpenoid with a novel skeleton, and compound 5 is a rare ent-abietane diterpenoid possessing a peroxide bridge. Compounds 1, 5-7, and 11-14 stimulate nerve growth factor mediated neurite outgrowth from PC12 cells.

Nerve Growth Factor Improves Survival and Function of Transplanted Islets Via TrkA-mediated β Cell Proliferation and Revascularization.

Nerve growth factor (NGF), which plays important roles in promoting growth and differentiation of nerve cells, has recently been reported as a regulator in pancreatic β cells in terms of insulin releasing function. In this study, we examined whether NGF stimulation would promote islet graft survival and function in islet transplantation. We found that supplementation of cultured islets with NGF improved the viability of islet cells and induced the production of insulin, vascular endothelial growth factor, and cellular proliferative markers. Because a specific inhibitor of TrkA, K252a, blocked all these effects, we propose that the TrkA receptor is the mediator of NGF stimulation. After transplantation to the kidney subcapsule and liver of syngenic diabetic mice, a higher rate of normoglycemic achievement, increased serum insulin, and improved glucose tolerance were observed in the mice transplanted with NGF-pretreated islet grafts. Histological analysis revealed higher expression of insulin and vascular endothelial growth factor, an increase in proliferative β cells, and revascularization in NGF-pretreated islet grafts without activation of any inflammatory cells. The NGF treatment can therefore serve as a new and promising therapeutic tool for improving islet graft viability and function in islet transplantation.

Differential regulation of macrophage phenotype by mature and pro-nerve growth factor.

To characterize the role of neurotrophin receptors on macrophages, we investigated the ability of nerve growth factor (NGF) and its precursor, proNGF, to regulate human macrophage phenotype. The p75 neurotrophin receptor (p75(NTR)) and TrkA were concentrated within overlapping domains on membrane ruffles. NGF stimulation of macrophages increased membrane ruffling, calcium spiking, phagocytosis and growth factor secretion. In contrast, proNGF induced podosome formation, increased migration, suppressed calcium spikes and increased neurotoxin secretion. These results demonstrate opposing roles of NGF and proNGF in macrophage regulation providing new avenues for pharmacological intervention during neuroinflammation.

Link N as a Therapeutic Agent to Treat Pain Associated with Intervertebral Disc Degeneration

Introduction The intervertebral discs (IVDs) are the largest avascular tissues in the body characterized by an abundant extracellular matrix and low cell density, with innervation limited at the most peripheral annular layers. During IVD degeneration, the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), can stimulate nerve growth and hyperinnervation of the inner annulus fibrosus (AF) and nucleus pulposus (NP), which further could lead to back pain. Back pain can potentially be suppressed by BMP4 supplementation to decrease the density of innervation. Link N is a naturally occurring peptide that can stimulate BMP4 in human IVDs. The aim of the present study was to determine the effect of Link N on NGF, BDNF, and substance P (SP) in human AF cells stimulated with cytokines or punctured bovine discs and determine the possible mechanisms related to its effect. Material and Methods Human lumbar spines were obtained through Héma-Québec harvesting organ donation within 24 hour after death. Annulus fibrosus (AF) cells were isolated from AF region of human IVDs with Thompson grade 2 of degeneration, cultured in monolayers and stimulated with TNFα (100 ng/mL) and IL1β (10 ng/mL) in the presence or absence of Link N (1 µg/mL) for 48 hours. Total RNA was isolated and gene expression of neurotrophins, substance P, and their receptors was measured using RT PCR. AF cells isolated from human IVDs with Thompson grade 4 were cultured for 48 hours with or without Link N (1 µg/mL) and the NGF release in the media was measured by Western blotting. Coccygeal IVDs from the tails of adult bovine steers (20–25 months) were isolated with the cartilaginous endplates. After 24 hours preconditioning in complete DMEM, the IVDs were treated (control, capsaicin [1.5 µg/mL), punctured by 16 G needle, punctured by 16 G needle and incubated with Link N [10 µg/mL]) and the disc culture media was collected at different time points for analysis. SP in the media was concentrated by solid phase extraction and was assayed by Elisa. Results Proinflammatory cytokines IL1β and TNFα have been shown to trigger the expression of NGF and SP, as well as promoting the degradation of the extracellular matrix in human IVD cells. Our results demonstrate that gene expression of NGF, BDNF, TrkA, and TrkB significantly decreased in human disc cells stimulated with either IL-1β or TNFα supplemented with Link N when compared with the control. Additionally, Link N was also able to inhibit NGF release in the media when AF grade 4 was stimulated with IL-1β. Link N supplementation also led to a significant decrease in SP after 24 hour in injured discs, when compared with controls. Conclusion Link N can suppress the stimulation of NGF, BDNF, and their receptors TrkA and TrkB, respectively, as well as the neurotransmitter SP in degenerate discs. This suggests that Link N has the potential to inhibit pain induced by neuronal innervation caused by disc degeneration. Thus, administration of Link N has the potential to not only repair the discs in early stages of the disease but also suppress pain.

Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation.

SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells using mass spectrometry-based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to TrkA. We showed that upon NGF stimulation, TrkA recruits the E3 ubiquitin ligase Cbl-b, which then becomes phosphorylated and ubiquitylated and decreases in abundance. We also found that recruitment of Cbl-b promotes TrkA ubiquitylation and degradation. Furthermore, the amount of phosphorylation of the kinase ERK and neurite outgrowth increased upon Cbl-b depletion in several neuroblastoma cell lines. Our findings suggest that Cbl-b limits NGF-TrkA signaling to control the length of neurites.

Open-channel microfluidic membrane device for long-term FT-IR spectromicroscopy of live adherent cells.

Spatially resolved infrared spectroscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal information on functional groups in biomolecules of a sample by their characteristic vibrational modes. One difficulty in performing long-term FT-IR measurements on live cells is the competition between the strong IR absorption from water and the need to supply nutrients and remove waste. In this proof of principle study, we developed an open-channel membrane device that allows long-term continuous IR measurement of live, adherent mammalian cells. Composed of a gold-coated porous membrane between a feeding channel and a viewing chamber, it allows cells to be maintained on the upper membrane surface in a thin layer of fluid while media is replenished from the feeding channel below. Using this device, we monitored the spatiotemporal chemical changes in living colonies of PC12 cells under nerve growth factor (NGF) stimulation for up to 7 days using both conventional globar and high-resolution synchrotron radiation-based IR sources. We identified the primary chemical change cells undergo is an increase in glycogen that may be associated with secretion of glycoprotein to protect the cells from evaporative stress at the air-liquid interface. Analyzing the spectral maps with multivariate methods of hierarchical cluster analysis (HCA) and principal component analysis (PCA), we found that the cells at the boundary of the colony and in a localized region in the center of the colony tend to produce more glycogen and glycoprotein than cells located elsewhere in the colony and that the degree of spatial heterogeneity decreases with time. This method provides a promising approach for long-term live-cell spectromicroscopy on mammalian cell systems.

Association of p75NTR and α9β1 integrin modulates NGF-dependent cellular responses

Direct interaction of α9β1 integrin with nerve growth factor (NGF) has been previously reported to induce pro-proliferative and pro-survival activities of non-neuronal cells. We investigated participation of p75NTR in α9β1 integrin-dependent cellular response to NGF stimulation. Using selective transfection of glioma cell lines with these receptors, we showed a strong, cation-independent association of α9 integrin subunit with p75NTR on the cellular membrane by selective immunoprecipitation experiments. The presence of the α9/p75NTR complex increases NGF-dependent cell adhesion, proliferation and migration. Other integrin subunits including β1 were not found in complex with p75NTR. FRET analysis indicated that p75NTR and α9 integrin subunit are not closely associated through their cytoplasmic domains, most probably because of the molecular interference with other cytoplasmic proteins such as paxillin. Interaction of α9β1 integrin with another ligand, VCAM-1 was not modulated by the p75NTR. α9/p75NTR complex elevated NGF-dependent activation of MAPK Erk1/2 arty for integrin that may create active complexes with other types of receptors belonging to the TNF superfamily.

The deubiquitinating enzyme UBPy/USP8 interacts with TrkA and inhibits neuronal differentiation in PC12 cells

The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPyC748A caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.

Neurotropin promotes NGF signaling through interaction of GM1 ganglioside with Trk neurotrophin receptor in PC12 cells

Activation of the high-affinity nerve growth factor (NGF) receptor Trk occurs through multiple processes consisted of translocation and clustering within the plasma membrane lipid rafts, dimerization and autophosphorylation. Here we found that a nonprotein extract of inflamed rabbit skin inoculated with vaccinia virus (Neurotropin®) enhanced efficiency of NGF signaling. In rat pheochromocytoma PC12 cells overexpressing Trk (PCtrk cells), Neurotropin augmented insufficient neurite outgrowth observed at suboptimal concentration of NGF (2ng/mL) in a manner depending on Trk kinase activity. Cellular exposure to Neurotropin resulted in an accumulation of Trk-GM1 complexes without affecting dimerization or phosphorylation states of Trk. Following NGF stimulation, Neurotropin significantly facilitated the time course of NGF-induced Trk autophosphorylation. These observations provide a unique mechanism controlling efficiency of NGF signaling, and raise the therapeutic potential of Neurotropin for various neurological conditions associated with neurotrophin dysfunction.

Nerve growth factor increases MMP9 activity in annulus fibrosus cells by upregulating lipocalin 2 expression.

Nerve growth factor (NGF) expression and activity is important in chronic lower back pain but may also act as a pro-catabolic factor in the pathogenesis of intervertebral disc (IVD) degeneration. Lipocalin 2 (Lcn2) expression in IVD was upregulated by NGF stimulation in our previous study. The current study was undertaken to identify potential mechanisms of the latter effect including potential interactions between Lcn2 and matrix metalloproteinase 9 (MMP9). Rat annulus fibrosus (AF) cells were stimulated by NGF and subjected to microarray analysis, subsequent real-time PCR, western immunoblotting, and immunofluorescence. Cells were treated with NGF in the absence or presence of the NGF inhibitor Ro 08-2750. Zymography and functional MMP9 assays were used to determine MMP9 activity, whilst the dimethyl-methylene blue assay was used to quantify the release of glycosaminoglycans (GAGs) reflecting catabolic effects following NGF treatment. Immunoprecipitation with immunoblotting was used to identify interactions between MMP9 and Lcn2. Increased expression of Lcn2 gene and protein following NGF stimulation was confirmed by microarray analysis, real-time PCR, western blot and immunofluorescence. Zymography showed that NGF enhanced 125-kDa gelatinase activity, identified as a Lcn2/MMP9 complex by immunoprecipitation and immunoblotting. Functional assays showed increased MMP9 activity and GAG release in the presence of NGF. The effects of NGF were neutralized by the presence of Ro 08-2750. NGF upregulates Lcn2 expression and increases MMP9 activity in AF cells; processes which are likely to potentiate degeneration of AF tissue in vivo. Anti-NGF treatment may have benefit for management of pain relief and slowing down progression of AF tissue degeneration.

Nicotine stimulates nerve growth factor in lung fibroblasts through an NFκB-dependent mechanism.

RationaleAirway hyperresponsiveness (AHR) is classically found in asthma, and persistent AHR is associated with poor asthma control. Although airway smooth muscle (ASM) cells play a critical pathophysiologic role in AHR, the paracrine contributions of surrounding cells such as fibroblasts to the contractile phenotype of ASM cells have not been examined fully. This study addresses the hypothesis that nicotine promotes a contractile ASM cell phenotype by stimulating fibroblasts to increase nerve growth factor (NGF) secretion into the environment.MethodsPrimary lung fibroblasts isolated from wild type and α7 nicotinic acetylcholine receptor (α7 nAChR) deficient mice were treated with nicotine (50 µg/ml) in vitro for 72 hours. NGF levels were measured in culture media and in bronchoalveolar lavage (BAL) fluid from asthmatic, smoking and non-smoking subjects by ELISA. The role of the NFκB pathway in nicotine-induced NGF expression was investigated by measuring NFκB nuclear translocation, transcriptional activity, chromatin immunoprecipitation assays, and si-p65 NFκB knockdown. The ability of nicotine to stimulate a fibroblast-mediated, contractile ASM cell phenotype was confirmed by examining expression of contractile proteins in ASM cells cultured with fibroblast-conditioned media or BAL fluid.ResultsNGF levels were elevated in the bronchoalveolar lavage fluid of nicotine-exposed mice, current smokers, and asthmatic children. Nicotine increased NGF secretion in lung fibroblasts in vitro in a dose-dependent manner and stimulated NFκB nuclear translocation, p65 binding to the NGF promoter, and NFκB transcriptional activity. These responses were attenuated in α7 nAChR deficient fibroblasts and in wild type fibroblasts following NFκB inhibition. Nicotine-treated, fibroblast-conditioned media increased expression of contractile proteins in ASM cells.ConclusionNicotine stimulates NGF release by lung fibroblasts through α7 nAChR and NFκB dependent pathways. These novel findings suggest that the nicotine-α7 nAChR-NFκB- NGF axis may provide novel therapeutic targets to attenuate tobacco smoke-induced AHR.

Abstract LB-328: SHP-1, p53 and Y674/Y675-phosphorylated-trkA: a molecular pathway and prognostic marker for neuroblastoma

Abstract Neuroblastomas, which have sympathoadrenergic origin, are the most frequent childhood extracranial tumors. Good prognosis tumors (favourable cytogenetics, differentiated) express nerve growth factor (NGF) receptor tyrosine kinase trkA, whereas poor prognosis neuroblastomas (adverse cytogenetics, Myc amplification, poor differentiation) express neurotrophin receptor tyrosine kinase trkB. On NGF stimulation, trkA is activated by tyrosine phosphorylation and outcome is cell/tissue-dependent, and can signal for differentiation. We have shown that in rat sympathoadrenergic PC12 cells, wild-type (wt)p53 expression induces trkA tyrosine phosphorylation and trkA dependent signalling, promoting their NGF independent differentiation. In breast cancer tumors and neuroblastomas, high trkA levels are associated with good prognosis. In both tumour types the p53 mutation incidence is 20-25%. The tyrosine phosphatase SHP-1 dephosphorylates trkA tyrosines 674/675(Y674/Y675). We have shown that in breast cancer wt-p53 represses SHP-1 expression and induces trkA-Y674/Y675 phosphorylation, leading to NGF-independent trkA activation. This signals to suppress breast cancer proliferation by promoting cell-cycle arrest. Furthermore, in breast cancer, coexpression of phosphorylated trkA-Y674/Y675, wt-p53 and low SHP-1 levels, correlates with prolonged 15 years disease free survival (DFS). Our experiments provide a mechanism for trkA expression correlation with good prognosis in breast cancer and emphasise the importance of this mechanism in cancer. As neuroblastomas contain different cell populations, it has been difficult to identify prognostic biomarkers that would predict patient outcome. We have shown that wt-p53 repression of SHP-1 leads to trkA-Y674/Y675 phosphorylation and neuroblastoma differentiation. Neuroblastoma differentiation is associated with improved DFS. Thus we asked if, the presence of wt-p53 and SHP-1 downregulation, together with phosphorylated trkA, in neuroblastoma samples, correlates with favourable outcome. Confirmation of this mechanism would validate new molecular targets for neuroblastoma. Neuroblastoma arrays containing 169 samples were stained for p53, SHP1 and phosphorylated trkA-Y674/Y675 expression. Multivariate analysis showed low p53 levels associated with 65% 5 years DFS (hazard-ratio (HR)=0.35, p=0.09), whereas, high SHP1 expression was associated with a worse DFS (HR=1.37, p=0.028). Phosphorylated trkA-Y674/Y675 expression correlated with 88% 5 years DFS (HR=0.12, (p=0.001)). As differentiation is associated with improved survival, the differentiation markers MAP2 and Vimentin were included. Expression of MAP2 and Vimentin correlated with good DFS (71% (HR=0.29, p=0.027) and 35% (HR=0.65, p=0.014)). Myc amplification correlated with reduced DFS (HR=4.84, p=0.001) as expected. Importantly, multivariate-analysis of patients with tumors harbouring p53 together with high SHP-1 levels and phosphorylated trkA-Y674/Y675 showed 69% 5 years DFS (HR=0.29, p=0.001). Favourable DFS was seen when MAP2 and Vimentin (HR=0.53, p=0.032 and HR=0.51, p=0035) were included. Further statistical analysis which considers tumor cytogenetics will be presented. Our results will allow refinement of the prognostic tools available for neuroblastoma with direct impact on child healthcare. Citation Format: Gehad Youssef, Cheryl Gillet, Dyanne Rampling, Mirza Chagtay, Alex Virasami, Neil Sebire, John Anderson, Ximena Montano. SHP-1, p53 and Y674/Y675-phosphorylated-trkA: a molecular pathway and prognostic marker for neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-328. doi:10.1158/1538-7445.AM2014-LB-328

Nerve growth factor promotes expression of novel genes in intervertebral disc cells that regulate tissue degradation: Laboratory investigation.

Increased neurotrophin activity in degenerative intervertebral discs (IVDs) is one potential cause of chronic low-back pain (LBP). The aim of the study was to assess if nerve growth factor (NGF) might alter gene expression of IVD cells and contribute to disc degeneration by enhancing expression or activity of factors that cause breakdown of IVD matrix. Rat-tail IVD cells were stimulated by NGF and subjected to microarray analysis. Real-time polymerase chain reaction, Western blotting, and immunocytochemistry of rat and human IVD cells and tissues treated with NGF in vitro in the absence or presence of the NGF inhibitor Ro 08-2750 were used to confirm findings of the microarray studies. Phosphorylation of mitogen-activated protein kinase (MAPK) was used to identify cell signaling pathways involved in NGF stimulation in the absence or presence of Ro 08-2750. Microarray analysis demonstrated increased expression of chitinase 3-like 1 (Chi3l1), lipocalin 2 (Lcn2), and matrix metalloproteinase-3 (Mmp3) following NGF stimulation of rat IVD cells in vitro. Increased gene expression was confirmed by real-time polymerase chain reaction with a relative increase in the Mmp/Timp ratio. Increased expression of Chi3l1, Lcn2, and Mmp3 following NGF stimulation was also demonstrated in rat cells and human tissue in vitro. Effects of NGF on protein expression were blocked by an NGF inhibitor and appear to function through the extracellular-regulation kinase 1/2 (ERK1/2) MAPK pathway. Nerve growth factor has potential effects on matrix turnover activity and influences the catabolic/anabolic balance of IVD cells in an adverse way that may potentiate IVD degeneration. Anti-NGF treatment might be beneficial to ameliorate progressive tissue breakdown in IVD degeneration and may lead to pain relief.

TrkA mediated translocation of TRPv1 receptor is reduced in afferent neurons of cadiomyopathic rats (1169.16)

Previous studies have suggested that the decreased activity of TRPv1 receptor, a vanilloid receptor, plays a role in the development of an exaggerated exercise pressor reflex observed in the cardiomyopathic (CM) rat model. A requirement of TRPv1 activity is its localization to the cell membrane, which is mediated by the TrkA receptor. To determine if the translocation of TRPv1 is impaired in CM rats, we compared the levels of TRPv1 in the cell membranes of neurons from dorsal root ganglia (DRG) from normal and CM rats. Cells were stained for TRPv1 and fluorescence intensities of membrane and total cell were quantified. Localization of TRPv1 was evaluated in the presence or absence of Nerve Growth Factor (NGF) stimulation, and with a TrkA inhibitor in the presence of NGF. NGF treated neurons from normal animals displayed an increased ratio of membrane to total cell (M:T) TRPv1 as compared to untreated control. The addition of a TrkA inhibitor returned M:T to the level of non‐stimulated neurons. In the neurons from CM rats, NGF failed to increase the M:T, and treatment with TrkA inhibitor also did not change M:T level. These results suggest that DRG from CM rats are defective in their ability to translocate TRPv1 to the membrane upon NGF stimulation and this occurs via the TrkA receptor pathway.Grant Funding Source: Supported by NIH 2R01HL070242‐07A2 (MGG)

Light-Mediated Kinetic Control Reveals the Temporal Effect of the Raf/MEK/ERK Pathway in PC12 Cell Neurite Outgrowth

It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network.

Protocol and cell responses in three-dimensional conductive collagen gel scaffolds with conductive polymer nanofibres for tissue regeneration.

It has been established that nerves and skeletal muscles respond and communicate via electrical signals. In regenerative medicine, there is current emphasis on using conductive nanomaterials to enhance electrical conduction through tissue-engineered scaffolds to increase cell differentiation and tissue regeneration. We investigated the role of chemically synthesized polyaniline (PANI) and poly(3,4-ethylenedioxythiophene) (PEDOT) conductive polymer nanofibres for conductive gels. To mimic a naturally derived extracellular matrix for cell growth, type I collagen gels were reconstituted with conductive polymer nanofibres and cells. Cell viability and proliferation of PC-12 cells and human skeletal muscle cells on these three-dimensional conductive collagen gels were evaluated in vitro. PANI and PEDOT nanofibres were found to be cytocompatible with both cell types and the best results (i.e. cell growth and gel electrical conductivity) were obtained with a low concentration (0.5 wt%) of PANI. After 7 days of culture in the conductive gels, the densities of both cell types were similar and comparable to collagen positive controls. Moreover, PC-12 cells were found to differentiate in the conductive hydrogels without the addition of nerve growth factor or electrical stimulation better than collagen control. Importantly, electrical conductivity of the three-dimensional gel scaffolds increased by more than 400% compared with control. The increased conductivity and injectability of the cell-laden collagen gels to injury sites in order to create an electrically conductive extracellular matrix makes these biomaterials very conducive for the regeneration of tissues.

Induction of nerve growth factor expression and release by mechanical and inflammatory stimuli in chondrocytes: possible involvement in osteoarthritis pain

IntroductionNerve growth factor (NGF) level is increased in osteoarthritis (OA) joints and is involved in pain associated with OA. Stimuli responsible for NGF stimulation in chondrocytes are unknown. We investigated whether mechanical stress and proinflammatory cytokines may influence NGF synthesis by chondrocytes.MethodsPrimary cultures of human OA chondrocytes, newborn mouse articular chondrocytes or cartilage explants were stimulated by increasing amounts of IL-1β, prostaglandin E2 (PGE2), visfatin/nicotinamide phosphoribosyltransferase (NAMPT) or by cyclic mechanical compression (0.5 Hz, 1 MPa). Before stimulation, chondrocytes were pretreated with indomethacin, Apo866, a specific inhibitor of NAMPT enzymatic activity, or transfected by siRNA targeting visfatin/NAMPT. mRNA NGF levels were assessed by real-time quantitative PCR and NGF released into media was determined by ELISA.ResultsUnstimulated human and mouse articular chondrocytes expressed low levels of NGF (19.2 ± 8.7 pg/mL, 13.5 ± 1.0 pg/mL and 4.4 ± 0.8 pg/mL/mg tissue for human and mouse articular chondrocytes and costal explants, respectively). Mechanical stress induced NGF release in conditioned media. When stimulated by IL-1β or visfatin/NAMPT, a proinflammatory adipokine produced by chondocytes in response to IL-1β, a dose-dependent increase in NGF mRNA expression and NGF release in both human and mouse chondrocyte conditioned media was observed. Visfatin/NAMPT is also an intracellular enzyme acting as the rate-limiting enzyme of the generation of NAD. The expression of NGF induced by visfatin/NAMPT was inhibited by Apo866, whereas IL-1β-mediated NGF expression was not modified by siRNA targeting visfatin/NAMPT. Interestingly, PGE2, which is produced by chondrocytes in response to IL-1β and visfatin/NAMPT, did not stimulate NGF production. Consistently, indomethacin, a cyclooxygenase inhibitor, did not counteract IL-1β-induced NGF production.ConclusionsThese results show that mechanical stress, IL-1β and extracellular visfatin/NAMPT, all stimulated the expression and release of NGF by chondrocytes and thus suggest that the overexpression of visfatin/NAMPT and IL-1β in the OA joint and the increased mechanical loading of cartilage may mediate OA pain via the stimulation of NGF expression and release by chondrocytes.

DNA damage and oxidative injury are associated with hypomyelination in the corpus callosum of newborn NbnCNS‐del mice

Nijmegen breakage syndrome (NBS), caused by mutation of the Nbn gene, is a recessive genetic disorder characterized by immunodeficiency, elevated sensitivity to ionizing radiation, chromosomal instability, microcephaly, and high predisposition to malignancies. To explore the underlying molecular mechanisms of NBS microcephaly, Frappart et al. previously inactivated Nbn gene in the central nervous system (CNS) of mice by the nestin-Cre targeting gene system and generated Nbn(CNS-del) mice. Here we first report that Nbn gene inactivation induces the defective proliferation and enhanced apoptosis of the oligodendrocyte precursor cells (OPCs), contributing to the severe hypomyelination of the nerve fibers of the corpus callosum. Under conditions of DNA damage and oxidative stress, the distinct regulatory roles of ATM-Chk2 signaling and AKT/mTOR signaling are responsible for the defective proliferation and enhanced apoptosis of the Nbn-deficient OPCs. In addition, specific HDAC isoforms may play distinctive roles in regulating the myelination of the Nbn-deficient OPCs. However, brain-derived neurotrophic factor and nerve growth factor stimulation attenuates the oxidative stress and thereby increases the proliferation of the Nbn-deficient OPCs, which is accompanied by upregulation of the AKT/mTOR/P70S6K signaling pathway. Taken together, these findings demonstrate that DNA damage and oxidative stress resulting from Nbn gene inactivation are associated with hypomyelination of the nerve fibers of corpus callosum.

APP Regulates NGF Receptor Trafficking and NGF-Mediated Neuronal Differentiation and Survival

β-amyloid precursor protein (APP) is a key factor in Alzheimer's disease (AD) but its physiological function is largely undetermined. APP has been found to regulate retrograde transport of nerve growth factor (NGF), which plays a crucial role in mediating neuronal survival and differentiation. Herein, we reveal the mechanism underlying APP-mediated NGF trafficking, by demonstrating a direct interaction between APP and the two NGF receptors, TrkA and p75NTR. Downregulation of APP leads to reduced cell surface levels of TrkA/p75NTR and increased endocytosis of TrkA/p75NTR and NGF. In addition, APP-deficient cells manifest defects in neurite outgrowth and are more susceptible to Aβ-induced neuronal death at physiological levels of NGF. However, APP-deficient cells show better responses to NGF-stimulated differentiation and survival than control cells. This may be attributed to increased receptor endocytosis and enhanced activation of Akt and MAPK upon NGF stimulation in APP-deficient cells. Together, our results suggest that APP mediates endocytosis of NGF receptors through direct interaction, thereby regulating endocytosis of NGF and NGF-induced downstream signaling pathways for neuronal survival and differentiation.