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Sample records for growth factor-induced neurite

  1. Inhibitory Activity of Yokukansankachimpihange against Nerve Growth Factor-Induced Neurite Growth in Cultured Rat Dorsal Root Ganglion Neurons.

    PubMed

    Murayama, Chiaki; Watanabe, Shimpei; Nakamura, Motokazu; Norimoto, Hisayoshi

    2015-01-01

    Chronic pruritus is a major and distressing symptom of many cutaneous diseases, however, the treatment remains a challenge in the clinic. The traditional Chinese-Japanese medicine (Kampo medicine) is a conservative and increasingly popular approach to treat chronic pruritus for both patients and medical providers. Yokukansankachimpihange (YKH), a Kampo formula has been demonstrated to be effective in the treatment of itching of atopic dermatitis in Japan although its pharmacological mechanism is unknown clearly. In an attempt to clarify its pharmacological actions, in this study, we focused on the inhibitory activity of YKH against neurite growth induced with nerve growth factor (NGF) in cultured rat dorsal root ganglion (DRG) neurons because epidermal hyperinnervation is deeply related to itch sensitization. YKH showed approximately 200-fold inhibitory activity against NGF-induced neurite growth than that of neurotropin (positive control), a drug used clinically for treatment of chronic pruritus. Moreover, it also found that Uncaria hook, Bupleurum root and their chemical constituents rhynchophylline, hirsutine, and saikosaponin a, d showed inhibitory activities against NGF-induced neurite growth, suggesting they should mainly contribute to the inhibitory activity of YKH. Further study on the effects of YKH against epidermal nerve density in "itch-scratch" animal models is under investigation. PMID:26287150

  2. Distinctive effect on nerve growth factor-induced PC12 cell neurite outgrowth by two unique neolignan enantiomers from Illicium merrillianum

    PubMed Central

    Tian, Xinhui; Yue, Rongcai; Zeng, Huawu; Li, Honglin; Shan, Lei; He, Weiwei; Shen, Yunheng; Zhang, Weidong

    2015-01-01

    Merrillianoid (1), a racemic neolignan possessing the characteristic benzo-2,7-dioxabicyclo[3.2.1]octane moiety, was isolated from the branches and leaves of Illicium merrillianum. Chiral separation of 1 gave two enantiomers (+)−1 and (−)−1. The structure of 1 was established by comprehensive spectroscopic analysis and single crystal X-ray diffraction. The absolute configurations of enantiomers were determined by quantum mechanical calculation. Compound (+)−1 exhibited a better neurotrophic activity than racemate 1 by promoting nerve growth factor (NGF) induced PC12 cell neurite outgrowth, while (−)−1 showed a distinctive inhibitory effect. Furthermore, a mechanism study indicated that the two enantiomers influenced NGF-induced neurite outgrowth of PC12 cells possibly by interacting with the trkA receptor, and extracellular signal regulated kinases 1/2 (ERK1/2) and mitogen-activated protein kinase (MEK) in Ras/ERK signal cascade. But the phosphorylation level of serine/threonine kinase Akt1 and Akt2 in PI3K/Akt signal pathway showed no significant difference between (+)−1 and (−)−1. PMID:26585042

  3. Distinctive effect on nerve growth factor-induced PC12 cell neurite outgrowth by two unique neolignan enantiomers from Illicium merrillianum

    NASA Astrophysics Data System (ADS)

    Tian, Xinhui; Yue, Rongcai; Zeng, Huawu; Li, Honglin; Shan, Lei; He, Weiwei; Shen, Yunheng; Zhang, Weidong

    2015-11-01

    Merrillianoid (1), a racemic neolignan possessing the characteristic benzo-2,7-dioxabicyclo[3.2.1]octane moiety, was isolated from the branches and leaves of Illicium merrillianum. Chiral separation of 1 gave two enantiomers (+)-1 and (-)-1. The structure of 1 was established by comprehensive spectroscopic analysis and single crystal X-ray diffraction. The absolute configurations of enantiomers were determined by quantum mechanical calculation. Compound (+)-1 exhibited a better neurotrophic activity than racemate 1 by promoting nerve growth factor (NGF) induced PC12 cell neurite outgrowth, while (-)-1 showed a distinctive inhibitory effect. Furthermore, a mechanism study indicated that the two enantiomers influenced NGF-induced neurite outgrowth of PC12 cells possibly by interacting with the trkA receptor, and extracellular signal regulated kinases 1/2 (ERK1/2) and mitogen-activated protein kinase (MEK) in Ras/ERK signal cascade. But the phosphorylation level of serine/threonine kinase Akt1 and Akt2 in PI3K/Akt signal pathway showed no significant difference between (+)-1 and (-)-1.

  4. 15d-prostaglandin J2 enhancement of nerve growth factor-induced neurite outgrowth is blocked by the chemoattractant receptor- homologous molecule expressed on T-helper type 2 cells (CRTH2) antagonist CAY10471 in PC12 cells.

    PubMed

    Hatanaka, Michiyoshi; Shibata, Norihiro; Shintani, Norihito; Haba, Ryota; Hayata, Atsuko; Hashimoto, Hitoshi; Baba, Akemichi

    2010-01-01

    The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is the most recently identified prostaglandin (PG) receptor for both PGD(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). We examined the mechanism by which 15d-PGJ(2) enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. CAY10471 (CRTH2 antagonist) inhibited both the neurite-promotion and p38 mitogen-activated protein (MAP) kinase phosphorylation induced by 15d-PGJ(2). In contrast, 13,14-dihydro-15-keto-PGD(2 )(DK-PGD(2)) (selective CRTH2 agonist) stimulated its phosphorylation but failed to produce neurite-promoting effects. These suggest, for the first time, the action of 15d-PGJ(2) is mediated by CRTH2, although the CRTH2 activation alone is insufficient for the underlying action. PMID:20424389

  5. Purines in neurite growth and astroglia activation.

    PubMed

    Heine, Claudia; Sygnecka, Katja; Franke, Heike

    2016-05-01

    The mammalian nervous system is a complex, functional network of neurons, consisting of local and long-range connections. Neuronal growth is highly coordinated by a variety of extracellular and intracellular signaling molecules. Purines turned out to be an essential component of these processes. Here, we review the current knowledge about the involvement of purinergic signaling in the regulation of neuronal development. We particularly focus on its role in neuritogenesis: the formation and extension of neurites. In the course of maturation mammals generally lose their ability to regenerate the central nervous system (CNS) e.g. after traumatic brain injury; although, spontaneous regeneration still occurs in the peripheral nervous system (PNS). Thus, it is crucial to translate the knowledge about CNS development and PNS regeneration into novel approaches to enable neurons of the mature CNS to regenerate. In this context we give a general overview of growth-inhibitory and growth-stimulatory factors and mechanisms involved in neurite growth. With regard to neuronal growth, astrocytes are an important cell population. They provide structural and metabolic support to neurons and actively participate in brain signaling. Astrocytes respond to injury with beneficial or detrimental reactions with regard to axonal growth. In this review we present the current knowledge of purines in these glial functions. Moreover, we discuss organotypic brain slice co-cultures as a model which retains neuron-glia interactions, and further presents at once a model for CNS development and regeneration. In summary, the purinergic system is a pivotal factor in neuronal development and in the response to injury. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'. PMID:26498067

  6. Preparation of embryonic retinal explants to study CNS neurite growth.

    PubMed

    Hanea, Sonia T; Shanmugalingam, Ushananthini; Fournier, Alyson E; Smith, Patrice D

    2016-05-01

    This protocol outlines the preparation of embryonic mouse retinal explants, which provides an effective technique to analyze neurite outgrowth in central nervous system (CNS) neurons. This validated ex vivo system, which displays limited neuronal death, is highly reproducible and particularly amenable to manipulation. Our previously published studies involving embryonic chick or adult mouse retinal explants were instrumental in the preparation of this protocol; aspects of these previous techniques were combined, adopted and optimized. This protocol thus permits more efficient analysis of neurite growth. Briefly, the retina is dissected from the embryonic mouse eye using precise techniques that take into account the small size of the embryonic eye. The approach applied ensures that the retinal ganglion cell (RGC) layer faces the adhesion substrate on coated cover slips. Neurite growth is clear, well-delineated and readily quantifiable. These retinal explants can therefore be used to examine the neurite growth effects elicited by potential therapeutic agents. PMID:27072342

  7. Serum- and substratum-dependent modulation of neuritic growth.

    PubMed

    Skaper, S D; Selak, I; Varon, S

    1983-01-01

    Explants of embryonic day 8 (E8) chicken dorsal root ganglia (DRG) have been cultured with medium containing serum or the serum-free supplement N1 on one of three substrata: collagen, polyornithine (PORN), or PORN exposed to a polyornithine-binding neurite-promoting factor (PNPF-PORN). Replicate cultures were maintained with or without nerve growth factor (NGF). NGF elicited its classical neuritic outgrowth on all three substrata in serum-containing or serum-free medium. In the absence of NGF, however, a gradation of increasing neurite growth was seen with: PNPF-PORN greater than PORN greater than collagen. This response occurred in both media. In addition, the neuritic halo in each instance was markedly more developed in the absence of serum, especially on PNPF-PORN. Nonneuronal behaviors reflected both serum and substratum influences: thus, nonneuronal outgrowth consisted mainly of flat cells with serum and collagen, was nonexistent with serum and PORN or PNPF-PORN, and involved mostly Schwann-like scattered cells in the absence of serum on any one substratum. The serum-dependent behaviors of ganglionic neurites were examined further with explants from chicken E11 sympathetic ganglia. A single substratum was used (PORN), without exogenous trophic factor. Neurite outgrowth was depressed by the presence of fetal calf serum, thus supporting the generality of this phenomenon. Lastly, PC12 cells, a clonal line of rat pheochromocytoma, will grow neurites in the presence of NGF after 48 hr in serum-free, but not serum-containing media. Addition of serum to serum-free cultures at this time results in the rapid and complete retraction of neurites. PMID:6876195

  8. Fetal calf serum-mediated inhibition of neurite growth from ciliary ganglion neurons in vitro.

    PubMed

    Davis, G E; Skaper, S D; Manthorpe, M; Moonen, G; Varon, S

    1984-01-01

    Embryonic chick ciliary ganglion (CG) neurons cultured in fetal calf serum-containing medium have been previously reported to extend neurites on polyornithine (PORN) substrata precoated with a neurite-promoting factor (PNPF) from rat schwannoma-conditioned medium. On PORN substrata alone, however, no neuritic growth occurred. This was interpreted as evidence that PORN was an incompetent substratum for ciliary neuritic growth. In this study, we now find that an untreated PORN substratum allows neuritic growth in serum-free defined medium. When PNPF was added to PORN, a more rapid and extensive neuritic response occurred. After 5 hr of culture, a 60% neuritic response occurred on PNPF/PORN, whereas no neurons initiated neurites until 10-12 hr on PORN. The inhibitory effect of fetal calf serum noted above on PORN could be obtained in part by pretreating the substratum with serum for 1 hr. Maximal inhibitory effects in the PORN pretreatment were achieved after 30 min and were not further improved by treatments up to 4 hr. Bovine serum albumin was also found to inhibit neurite growth on PORN to about 60% of the inhibition obtained by an equivalent amount of serum protein. Fetal calf serum was shown to cause a 15% reduction in the percentage of neurons bearing neurites after its addition to 18-hr serum-free PORN cultures and to cause statistically significant reductions in neurite lengths measured 2 hr later. PMID:6481819

  9. Insulin signaling regulates neurite growth during metamorphic neuronal remodeling

    PubMed Central

    Gu, Tingting; Zhao, Tao; Hewes, Randall S.

    2014-01-01

    Summary Although the growth capacity of mature neurons is often limited, some neurons can shift through largely unknown mechanisms from stable maintenance growth to dynamic, organizational growth (e.g. to repair injury, or during development transitions). During insect metamorphosis, many terminally differentiated larval neurons undergo extensive remodeling, involving elimination of larval neurites and outgrowth and elaboration of adult-specific projections. Here, we show in the fruit fly, Drosophila melanogaster (Meigen), that a metamorphosis-specific increase in insulin signaling promotes neuronal growth and axon branching after prolonged stability during the larval stages. FOXO, a negative effector in the insulin signaling pathway, blocked metamorphic growth of peptidergic neurons that secrete the neuropeptides CCAP and bursicon. RNA interference and CCAP/bursicon cell-targeted expression of dominant-negative constructs for other components of the insulin signaling pathway (InR, Pi3K92E, Akt1, S6K) also partially suppressed the growth of the CCAP/bursicon neuron somata and neurite arbor. In contrast, expression of wild-type or constitutively active forms of InR, Pi3K92E, Akt1, Rheb, and TOR, as well as RNA interference for negative regulators of insulin signaling (PTEN, FOXO), stimulated overgrowth. Interestingly, InR displayed little effect on larval CCAP/bursicon neuron growth, in contrast to its strong effects during metamorphosis. Manipulations of insulin signaling in many other peptidergic neurons revealed generalized growth stimulation during metamorphosis, but not during larval development. These findings reveal a fundamental shift in growth control mechanisms when mature, differentiated neurons enter a new phase of organizational growth. Moreover, they highlight strong evolutionarily conservation of insulin signaling in neuronal growth regulation. PMID:24357229

  10. Applied electric field enhances DRG neurite growth: influence of stimulation media, surface coating and growth supplements

    NASA Astrophysics Data System (ADS)

    Wood, Matthew D.; Willits, Rebecca Kuntz

    2009-08-01

    Electrical therapies have been found to aid repair of nerve injuries and have been shown to increase and direct neurite outgrowth during stimulation. This enhanced neural growth existed even after the electric field (EF) or stimulation was removed, but the factors that may influence the enhanced growth, such as stimulation media or surface coating, have not been fully investigated. This study characterized neurite outgrowth and branching under various conditions: EF magnitude and application time, ECM surface coating, medium during EF application and growth supplements. A uniform, low-magnitude EF (24 or 44 V m-1) was applied to dissociated chick embryo dorsal root ganglia seeded on collagen or laminin-coated surfaces. During the growth period, cells were either exposed to NGF or N2, and during stimulation cells were exposed to either unsupplemented media (Ca2+) or PBS (no Ca2+). Parallel controls for each experiment included cells exposed to the chamber with no stimulation and cells remaining outside the chamber. After brief electrical stimulation (10 min), neurite length significantly increased 24 h after application for all conditions studied. Of particular interest, increased stimulation time (10-100 min) further enhanced neurite length on laminin but not on collagen surfaces. Neurite branching was not affected by stimulation on any surface, and no preferential growth of neurites was noted after stimulation. Overall, the results of this report suggest that short-duration electric stimulation is sufficient to enhance neurite length under a variety of conditions. While further data are needed to fully elucidate a mechanism for this increased growth, these data suggest that one focus of those investigations should be the interaction between the growth cone and the substrata.

  11. Applied electric field enhances DRG neurite growth: influence of stimulation media, surface coating and growth supplements.

    PubMed

    Wood, Matthew D; Willits, Rebecca Kuntz

    2009-08-01

    Electrical therapies have been found to aid repair of nerve injuries and have been shown to increase and direct neurite outgrowth during stimulation. This enhanced neural growth existed even after the electric field (EF) or stimulation was removed, but the factors that may influence the enhanced growth, such as stimulation media or surface coating, have not been fully investigated. This study characterized neurite outgrowth and branching under various conditions: EF magnitude and application time, ECM surface coating, medium during EF application and growth supplements. A uniform, low-magnitude EF (24 or 44 V m(-1)) was applied to dissociated chick embryo dorsal root ganglia seeded on collagen or laminin-coated surfaces. During the growth period, cells were either exposed to NGF or N2, and during stimulation cells were exposed to either unsupplemented media (Ca(2+)) or PBS (no Ca(2+)). Parallel controls for each experiment included cells exposed to the chamber with no stimulation and cells remaining outside the chamber. After brief electrical stimulation (10 min), neurite length significantly increased 24 h after application for all conditions studied. Of particular interest, increased stimulation time (10-100 min) further enhanced neurite length on laminin but not on collagen surfaces. Neurite branching was not affected by stimulation on any surface, and no preferential growth of neurites was noted after stimulation. Overall, the results of this report suggest that short-duration electric stimulation is sufficient to enhance neurite length under a variety of conditions. While further data are needed to fully elucidate a mechanism for this increased growth, these data suggest that one focus of those investigations should be the interaction between the growth cone and the substrata. PMID:19494423

  12. Interleukin-1 beta and neurotrophin-3 synergistically promote neurite growth in vitro.

    PubMed

    Boato, Francesco; Hechler, Daniel; Rosenberger, Karen; Lüdecke, Doreen; Peters, Eva M; Nitsch, Robert; Hendrix, Sven

    2011-01-01

    Pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) are considered to exert detrimental effects during brain trauma and in neurodegenerative disorders. Consistently, it has been demonstrated that IL-1β suppresses neurotrophin-mediated neuronal cell survival rendering neurons vulnerable to degeneration. Since neurotrophins are also well known to strongly influence axonal plasticity, we investigated here whether IL-1β has a similar negative impact on neurite growth. We analyzed neurite density and length of organotypic brain and spinal cord slice cultures under the influence of the neurotrophins NGF, BDNF, NT-3 and NT-4. In brain slices, only NT-3 significantly promoted neurite density and length. Surprisingly, a similar increase of neurite growth was induced by IL-1β. Additionally, both factors increased the number of brain slices displaying maximal neurite growth. Furthermore, the co-administration of IL-1β and NT-3 significantly increased the number of brain slices displaying maximal neurite growth compared to single treatments. These data indicate that these two factors synergistically stimulate two distinct aspects of neurite outgrowth, namely neurite density and neurite length from acute organotypic brain slices. PMID:22200088

  13. Interleukin-1 beta and neurotrophin-3 synergistically promote neurite growth in vitro

    PubMed Central

    2011-01-01

    Pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) are considered to exert detrimental effects during brain trauma and in neurodegenerative disorders. Consistently, it has been demonstrated that IL-1β suppresses neurotrophin-mediated neuronal cell survival rendering neurons vulnerable to degeneration. Since neurotrophins are also well known to strongly influence axonal plasticity, we investigated here whether IL-1β has a similar negative impact on neurite growth. We analyzed neurite density and length of organotypic brain and spinal cord slice cultures under the influence of the neurotrophins NGF, BDNF, NT-3 and NT-4. In brain slices, only NT-3 significantly promoted neurite density and length. Surprisingly, a similar increase of neurite growth was induced by IL-1β. Additionally, both factors increased the number of brain slices displaying maximal neurite growth. Furthermore, the co-administration of IL-1β and NT-3 significantly increased the number of brain slices displaying maximal neurite growth compared to single treatments. These data indicate that these two factors synergistically stimulate two distinct aspects of neurite outgrowth, namely neurite density and neurite length from acute organotypic brain slices. PMID:22200088

  14. Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.

    PubMed

    Krug, Anne K; Balmer, Nina V; Matt, Florian; Schönenberger, Felix; Merhof, Dorit; Leist, Marcel

    2013-12-01

    Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants. PMID:23670202

  15. Neurite outgrowth at the interface of 2D and 3D growth environments

    NASA Astrophysics Data System (ADS)

    Kofron, Celinda M.; Fong, Vivian J.; Hoffman-Kim, Diane

    2009-02-01

    Growing neurons navigate complex environments, but in vitro systems for studying neuronal growth typically limit the cues to flat surfaces or a single type of cue, thereby limiting the resulting growth. Here we examined the growth of neurons presented with two-dimensional (2D) substrate-bound cues when these cues were presented in conjunction with a more complex three-dimensional (3D) architecture. Dorsal root ganglia (DRG) explants were cultured at the interface between a collagen I matrix and a glass coverslip. Laminin (LN) or chondroitin sulfate proteoglycans (CSPG) were uniformly coated on the surface of the glass coverslip or patterned in 50 µm tracks by microcontact printing. Quantitative analysis of neurite outgrowth with a novel grid system at multiple depths in the gel revealed several interesting trends. Most of the neurites extended at the surface of the gel when LN was presented whereas more neurites extended into the gel when CSPG was presented. Patterning of cues did not affect neurite density or depth of growth. However, neurite outgrowth near the surface of the gel aligned with LN patterns, and these extensions were significantly longer than neurites extended in other cultures. In interface cultures, DRG growth patterns varied with the type of cue where neurite density was higher in cultures presenting LN than in cultures presenting CSPG. These results represent an important step toward understanding how neurons integrate local structural and chemical cues to make net growth decisions.

  16. Growth, collapse, and stalling in a mechanical model for neurite motility

    NASA Astrophysics Data System (ADS)

    Recho, Pierre; Jerusalem, Antoine; Goriely, Alain

    2016-03-01

    Neurites, the long cellular protrusions that form the routes of the neuronal network, are capable of actively extending during early morphogenesis or regenerating after trauma. To perform this task, they rely on their cytoskeleton for mechanical support. In this paper, we present a three-component active gel model that describes neurites in the three robust mechanical states observed experimentally: collapsed, static, and motile. These states arise from an interplay between the physical forces driven by growth of the microtubule-rich inner core of the neurite and the acto-myosin contractility of its surrounding cortical membrane. In particular, static states appear as a mechanical traction or compression balance of these two parallel structures. The model predicts how the response of a neurite to a towing force depends on the force magnitude and recovers the response of neurites to several drug treatments that modulate the cytoskeleton active and passive properties.

  17. Changing growth of neurites of sensory ganglion by terahertz radiation

    NASA Astrophysics Data System (ADS)

    Tsurkan, M. V.; Smolyanskaya, O. A.; Bespalov, V. G.; Penniyainen, V. A.; Kipenko, A. V.; Lopatina, E. V.; Krylov, B. V.

    2012-02-01

    Application of terahertz radiation for the creation of medical equipment and solving of biological problems has become widely spread. From this point of view, the influence of THz radiation on the nerve fibers is of primary concern. In addition, several studies indicated both stimulating and depressive effects on nerve cells. However, the mechanism of this effect has not yet been studied, including the dose and exposure time. Our research was devoted to the impact of broadband pulsed THz radiation in the frequency range of 0.05 to 2 THz on the neurite growth in the sensory ganglia of 10-12-day chicken embryos. Dependence of changes in functional responses of cells on the average output power has been found. An increase in the stimulating effect was observed at the lowest power density used (0.5 μW/cm2). Through non-destructive process and choosing the correct parameters of THz radiation, potential control of neural response becomes possible, which can subsequently lead to new medical treatments.

  18. Survival and neurite growth of chick embryo spinal cord cells in serum-free culture.

    PubMed

    Tanaka, H; Obata, K

    1982-07-01

    Cell survival and neurite growth were investigated in serum-free spinal cord cell cultures on polyornithine coating (PORN). Cells were obtained from 6- or 7-day-old chick embryos. Isolated spinal cord cells required promoting factors for their survival and neurite growth. The survival-promoting factors were initially present in spinal cord cells. High density cultures, co-cultures with spinal cord explants, and spinal cord extract promoted survival of isolated spinal cord cells in MEM with no additives. Other tissue extracts (brain, liver, heart and skeletal muscle), serum, and serum-free conditioned medium (SF-CM) of muscle or glioma C6 cells also promoted survival. The active substances in the brain extract and SF-CM were shown to be protein and were separated into 3 fractions (approximately molecular weight 150,000, 70,000, 40,000) by gel filtration chromatography. Survival and neurite growth were suggested to be promoted by different factors because: (1) survival was promoted by both tissue extract and SF-CM, but neurite growth was promoted only by SF-CM; (2) the neurite growth-stimulating activity of SF-CM was lost following dialysis and heat (100 degrees C, 2 min) treatment; however, the survival-promoting activity was not. It was also suggested that spinal cord cells produce neurite growth promoting factors, but did not initially contain these factors. PMID:7104764

  19. Design of three-dimensional engineered protein hydrogels for tailored control of neurite growth.

    PubMed

    Lampe, Kyle J; Antaris, Alexander L; Heilshorn, Sarah C

    2013-03-01

    The design of bioactive materials allows tailored studies probing cell-biomaterial interactions, however, relatively few studies have examined the effects of ligand density and material stiffness on neurite growth in three-dimensions. Elastin-like proteins (ELPs) have been designed with modular bioactive and structural regions to enable the systematic characterization of design parameters within three-dimensional (3-D) materials. To promote neurite out-growth and better understand the effects of common biomaterial design parameters on neuronal cultures we here focused on the cell-adhesive ligand density and hydrogel stiffness as design variables for ELP hydrogels. With the inherent design freedom of engineered proteins these 3-D ELP hydrogels enabled decoupled investigations into the effects of biomechanics and biochemistry on neurite out-growth from dorsal root ganglia. Increasing the cell-adhesive RGD ligand density from 0 to 1.9×10(7)ligands μm(-3) led to a significant increase in the rate, length, and density of neurite out-growth, as quantified by a high throughput algorithm developed for dense neurite analysis. An approximately two-fold improvement in total neurite out-growth was observed in materials with the higher ligand density at all time points up to 7 days. ELP hydrogels with initial elastic moduli of 0.5, 1.5, or 2.1kPa and identical RGD ligand densities revealed that the most compliant materials led to the greatest out-growth, with some neurites extending over 1800μm by day 7. Given the ability of ELP hydrogels to efficiently promote neurite out-growth within defined and tunable 3-D microenvironments these materials may be useful in developing therapeutic nerve guides and the further study of basic neuron-biomaterial interactions. PMID:23128159

  20. Oxytocin Increases Neurite Length and Expression of Cytoskeletal Proteins Associated with Neuronal Growth.

    PubMed

    Lestanova, Z; Bacova, Z; Kiss, A; Havranek, T; Strbak, V; Bakos, J

    2016-06-01

    Neuropeptide oxytocin acts as a growth and differentiation factor; however, its effects on neurite growth are poorly understood. The aims of the present study were (1) to evaluate time effects of oxytocin on expression of nestin and MAP2; (2) to measure the effect of oxytocin on gene expression of β-actin, vimentin, cofilin, and drebrin; and (3) to measure changes in neurite length and number in response to oxytocin/oxytocin receptor antagonist L-371,257. Exposure of SH-SY5Y cells to 1 μM oxytocin resulted in a significant increase in gene expression and protein levels of nestin after 12, 24, and 48 h. Oxytocin treatment induced no changes in gene expression of MAP2; however, a decrease of protein levels was observed in all time intervals. Gene expression of β-actin, vimentin, and drebrin increased in response to oxytocin. Oxytocin induced significant elongation of neurites after 12, 24, and 48 h. No change in neurite length was observed in the presence of the combination of retinoic acid and oxytocin receptor antagonist L-371,257. Oxytocin treatment for 12 h increased the number of neurites. Overall, the present data suggest that oxytocin contributes to the regulation of expression of cytoskeletal proteins associated with growth of neuronal cones and induces neurite elongation mediated by oxytocin receptors at least in certain types of neuronal cells. PMID:26474566

  1. Enhanced Neurite Growth from Mammalian Neurons in Three-Dimensional Salmon Fibrin Gels

    PubMed Central

    Ju, Yo-El; Janmey, Paul A.; McCormick, Margaret; Sawyer, Evelyn S.; Flanagan, Lisa A.

    2007-01-01

    Three-dimensional fibrin matrices have been used as cellular substrates in vitro and as bridging materials for central nervous system repair. Cells can be embedded within fibrin gels since the polymerization process is non-toxic, making fibrin an attractive scaffold for transplanted cells. Most studies have utilized fibrin prepared from human or bovine blood proteins. However, fish fibrin may be well suited for neuronal growth since fish undergo remarkable central nervous system regeneration and molecules implicated in this process are present in fibrin. We assessed the growth of mammalian central nervous system neurons in bovine, human, and salmon fibrin and found that salmon fibrin gels encouraged the greatest degree of neurite (dendrite and axon) growth and were the most resistant to degradation by cellular proteases. The neurite growth-promoting effect was not due to the thrombin used to polymerize the gels or to any copurifying plasminogen. Co-purified fibronectin partially accounted for the effect on neurites, and blockade of fibrinogen/fibrin-binding integrins markedly decreased neurite growth. Anion exchange chromatography revealed different elution profiles for salmon and mammalian fibrinogens. These data demonstrate that salmon fibrin encourages the growth of neurites from mammalian neurons and suggest that salmon fibrin may be a beneficial scaffold for neuronal regrowth after CNS injury. PMID:17258313

  2. Polyester with Pendent Acetylcholine-Mimicking Functionalities Promotes Neurite Growth.

    PubMed

    Wang, Shaofei; Jeffries, Eric; Gao, Jin; Sun, Lijie; You, Zhengwei; Wang, Yadong

    2016-04-20

    Successful regeneration of nerves can benefit from biomaterials that provide a supportive biochemical and mechanical environment while also degrading with controlled inflammation and minimal scar formation. Herein, we report a neuroactive polymer functionalized by covalent attachment of the neurotransmitter acetylcholine (Ach). The polymer was readily synthesized in two steps from poly(sebacoyl diglyceride) (PSeD), which previously demonstrated biocompatibility and biodegradation in vivo. Distinct from prior acetylcholine-biomimetic polymers, PSeD-Ach contains both quaternary ammonium and free acetyl moieties, closely resembling native acetylcholine structure. The polymer structure was confirmed via (1)H nuclear magnetic resonance and Fourier-transform infrared spectroscopy. Hydrophilicity, charge, and thermal properties of PSeD-Ach were determined by tensiometer, zetasizer, differential scanning calorimetry, and thermal gravimetric analysis, respectively. PC12 cells exhibited the greatest proliferation and neurite outgrowth on PSeD-Ach and laminin substrates, with no significant difference between these groups. PSeD-Ach yielded much longer neurite outgrowth than the control polymer containing ammonium but no the acetyl group, confirming the importance of the entire acetylcholine-like moiety. Furthermore, PSeD-Ach supports adhesion of primary rat dorsal root ganglions and subsequent neurite sprouting and extension. The sprouting rate is comparable to the best conditions from previous report. Our findings are significant in that they were obtained with acetylcholine-like functionalities in 100% repeating units, a condition shown to yield significant toxicity in prior publications. Moreover, PSeD-Ach exhibited favorable mechanical and degradation properties for nerve tissue engineering application. Humidified PSeD-Ach had an elastic modulus of 76.9 kPa, close to native neural tissue, and could well recover from cyclic dynamic compression. PSeD-Ach showed a gradual in

  3. Inhibiting geranylgeranylation increases neurite branching and differentially activates cofilin in cell bodies and growth cones.

    PubMed

    Samuel, Filsy; Reddy, Jairus; Kaimal, Radhika; Segovia, Vianey; Mo, Huanbiao; Hynds, DiAnna L

    2014-08-01

    Inhibitors of the mevalonate pathway, including the highly prescribed statins, reduce the production of cholesterol and isoprenoids such as geranylgeranyl pyrophosphates. The Rho family of small guanine triphosphatases (GTPases) requires isoprenylation, specifically geranylgeranylation, for activation. Because Rho GTPases are primary regulators of actin filament rearrangements required for process extension, neurite arborization, and synaptic plasticity, statins may affect cognition or recovery from nervous system injury. Here, we assessed how manipulating geranylgeranylation affects neurite initiation, elongation, and branching in neuroblastoma growth cones. Treatment with the statin, lovastatin (20 μM), decreased measures of neurite initiation by 17.0 to 19.0 % when a source of cholesterol was present and increased neurite branching by 4.03- to 9.54-fold (regardless of exogenous cholesterol). Neurite elongation was increased by treatment with lovastatin only in cholesterol-free culture conditions. Treatment with lovastatin decreased growth cone actin filament content by up to 24.3 %. In all cases, co-treatment with the prenylation precursor, geranylgeraniol (10 μM), reversed the effect of lovastatin. In a prior work, statin effects on outgrowth were linked to modulating the actin depolymerizing factor, cofilin. In our assays, treatment with lovastatin or geranylgeraniol decreased cofilin phosphorylation in whole cell lysates. However, lovastatin increased cofilin phosphorylation in cell bodies and decreased it in growth cones, indicating differential regulation in specific cell regions. Together, we interpret these data to suggest that protein geranylgeranylation likely regulates growth cone actin filament content and subsequent neurite outgrowth through mechanisms that also affect actin nucleation and polymerization. PMID:24515839

  4. Increase in Growth Cone Size Correlates with Decrease in Neurite Growth Rate

    PubMed Central

    Ren, Yuan

    2016-01-01

    Several important discoveries in growth cone cell biology were made possible by the use of growth cones derived from cultured Aplysia bag cell neurons, including the characterization of the organization and dynamics of the cytoskeleton. The majority of these Aplysia studies focused on large growth cones induced by poly-L-lysine substrates at early stages in cell culture. Under these conditions, the growth cones are in a steady state with very little net advancement. Here, we offer a comprehensive cellular analysis of the motile behavior of Aplysia growth cones in culture beyond this pausing state. We found that average growth cone size decreased with cell culture time whereas average growth rate increased. This inverse correlation of growth rate and growth cone size was due to the occurrence of large growth cones with a peripheral domain larger than 100 μm2. The large pausing growth cones had central domains that were less consistently aligned with the direction of growth and could be converted into smaller, faster-growing growth cones by addition of a three-dimensional collagen gel. We conclude that the significant lateral expansion of lamellipodia and filopodia as observed during these culture conditions has a negative effect on neurite growth. PMID:27274874

  5. Controlled neuronal cell patterning and guided neurite growth on micropatterned nanofiber platforms

    NASA Astrophysics Data System (ADS)

    Malkoc, Veysi; Gallego-Perez, Daniel; Nelson, Tyler; Lannutti, John J.; Hansford, Derek J.

    2015-12-01

    Patterning neuronal cells and guiding neurite growth are important for applications such as prosthetics, cell based biosensors, and tissue engineering. In this paper, a microdevice is presented that provides neuronal cell patterning and guided neurite growth on a collagen coated gelatin/PCL nanofiber mat. The pattern consisted of a grid of polystyrene microwells/nodes to confine the cell bodies and orthogonal grooves to guide neurite growth from each node. Vacuum assisted cell seeding was used to localize cell bodies in the microwells and physically separate the cells during seeding. The electrospun nanofiber mats under the polystyrene microstructures were coated with collagen to enhance the cellular attachment and enhance differentiation. We evaluated the performance of our device using adhesion, viability, and differentiation assays of neuron-like PC12 cells compared to controls for vacuum seeding, spatial isolation and guidance, and collagen coating of the fibers. The device provided PC12 cell patterning with increased adhesion, differentiation, and guided neurite outgrowth compared to controls, demonstrating its potential for in vitro neuronal cell patterning studies.

  6. Effects of nerve growth factor and heart cell conditioned medium on neurite regeneration of aged sympathetic neurons in culture.

    PubMed

    Uchida, Y; Tomonaga, M

    1985-11-25

    The effects of nerve growth factor (NGF) and heart-cell-conditioned medium (HCM) on the neurite regeneration of aged sympathetic neurons were investigated in culture. Investigation of HCM was carried out by two different methods: one was the use of whole HCM on collagen substratum, which reflected component(s) effective in solution (HCM-S); the other was the use of polyornithine (PORN)-binding component(s) (P-HCM). Superior cervical ganglion neurons prepared from male mice from 6 to 30 months of age were cultured in MEM-10% FCS on collagen or gelatin-PORN substratum for 3 days. The number of neurons with neurites and the length of neurites were quantified as neurite production and elongation, respectively. Neuronal survival was not affected by addition of NGF, HCM-S or P-HCM. Neurite production of early adult neurons was enhanced by NGF, HCM-S or P-HCM. In contrast, neurite production of aged neurons was enhanced by only HCM-S, but not NGF or P-HCM. HCM-S did not promote neurite elongation in neurons at any age. Neurite elongation of early adult neurons was enhanced by NGF or P-HCM. Neurite elongation of aged neurons was enhanced by P-HCM. However, responsiveness of NGF for neurite elongation varied according to substrata. No age-related difference was found in neurite production and elongation in the absence of NGF, HCM-S or P-HCM. These results indicate that responsiveness of aged sympathetic neurons is various in different growth factors. PMID:3840716

  7. Anillin Regulates Neuronal Migration and Neurite Growth by Linking RhoG to the Actin Cytoskeleton.

    PubMed

    Tian, Dong; Diao, Min; Jiang, Yuxiang; Sun, Lingfei; Zhang, Yan; Chen, Zhucheng; Huang, Shanjin; Ou, Guangshuo

    2015-05-01

    Neuronal migration and neurite growth are essential events in neural development, but it remains unclear how guidance cues are transduced through receptors to the actin cytoskeleton, which powers these processes. We report that a cytokinetic scaffold protein, Anillin, is redistributed to the leading edge of the C. elegans Q neuroblast during cell migration and neurite growth. To bypass the requirement for Anillin in cytokinesis, we used the somatic CRISPR-Cas9 technique to generate conditional mutations in Anillin. We demonstrate that Anillin regulates cell migration and growth cone extension by stabilizing the F-actin network at the leading edge. Our biochemical analysis shows that the actin-binding domain of Anillin is sufficient to stabilize F-actin by antagonizing the F-actin severing activity of Cofilin. We further uncover that the active form of RhoG/MIG-2 directly binds to Anillin and recruits it to the leading edge. Our results reveal a novel pathway in which Anillin transduces the RhoG signal to the actin cytoskeleton during neuronal migration and neurite growth. PMID:25843030

  8. Regulation of Neurite Growth by Inorganic Pyrophosphatase 1 via JNK Dephosphorylation

    PubMed Central

    Tezuka, Yu; Okada, Mizuki; Tada, Yuka; Yamauchi, Junji; Nishigori, Hideo; Sanbe, Atsushi

    2013-01-01

    Neural cell differentiation during development is controlled by multiple signaling pathways, in which protein phosphorylation and dephosphorylation play an important role. In this study, we examined the role of pyrophosphatase1 (PPA1) in neuronal differentiation using the loss and gain of function analysis. Neuronal differentiation induced by external factors was studied using a mouse neuroblastoma cell line (N1E115). The neuronal like differentiation in N1E115 cells was determined by morphological analysis based on neurite growth length. In order to analyze the loss of the PPA1 function in N1E115, si-RNA specifically targeting PPA1 was generated. To study the effect of PPA1 overexpression, an adenoviral gene vector containing the PPA1 gene was utilized to infect N1E115 cells. To address the need for pyrophosphatase activity in PPA1, D117A PPA1, which has inactive pyrophosphatase, was overexpressed in N1E115 cells. We used valproic acid (VPA) as a neuronal differentiator to examine the effect of PPA1 in actively differentiated N1E115 cells. Si-PPA1 treatment reduced the PPA1 protein level and led to enhanced neurite growth in N1E115 cells. In contrast, PPA1 overexpression suppressed neurite growth in N1E115 cells treated with VPA, whereas this effect was abolished in D117A PPA1. PPA1 knockdown enhanced the JNK phosphorylation level, and PPA1 overexpression suppressed it in N1E115 cells. It seems that recombinant PPA1 can dephosphorylate JNK while no alteration of JNK phosphorylation level was seen after treatment with recombinant PPA1 D117A. Enhanced neurite growth by PPA1 knockdown was also observed in rat cortical neurons. Thus, PPA1 may play a role in neuronal differentiation via JNK dephosphorylation. PMID:23626709

  9. Effects of a synthetic bioactive peptide on neurite growth and nerve growth factor release in chondroitin sulfate hydrogels.

    PubMed

    Conovaloff, Aaron W; Beier, Brooke L; Irazoqui, Pedro P; Panitch, Alyssa

    2011-01-01

    Previous work has revealed robust dorsal root ganglia neurite growth in hydrogels of chondroitin sulfate. In the current work, it was determined whether addition of a synthetic bioactive peptide could augment neurite growth in these matrices via enhanced binding and sequestering of growth factors. Fluorescence recovery after photobleaching studies revealed that addition of peptide slowed nerve growth factor diffusivity in chondroitin sulfate gels, but not in control gels of hyaluronic acid. Furthermore, cultures of chick dorsal root ganglia in gels of hyaluronic acid or chondroitin sulfate revealed enhanced growth in chondroitin sulfate gels only upon addition of peptide. Taken together, these results suggest a synergistic nerve growth factor-binding activity between this peptide and chondroitin sulfate. PMID:23507745

  10. Microtopographical features generated by photopolymerization recruit RhoA/ROCK through TRPV1 to direct cell and neurite growth

    PubMed Central

    Li, Shufeng; Tuft, Bradley; Xu, Linjing; Polacco, Marc; Clarke, Joseph C.; Guymon, C. Allan; Hansen, Marlan R.

    2015-01-01

    Cell processes, including growth cones, respond to biophysical cues in their microenvironment to establish functional tissue architecture and intercellular networks. The mechanisms by which cells sense and translate biophysical cues into directed growth are unknown. We used photopolymerization to fabricate methacrylate platforms with patterned microtopographical features that precisely guide neurite growth and Schwann cell alignment. Pharmacologic inhibition of the transient receptor potential cation channel subfamily V member 1 (TRPV1) or reduced expression of TRPV1 by RNAi significantly disrupts neurite guidance by these microtopographical features. Exogenous expression of TRPV1 induces alignment of NIH3T3 fibroblasts that fail to align in the absence of TRPV1, further implicating TRPV1 channels as critical mediators of cellular responses to biophysical cues. Microtopographic features increase RhoA activity in growth cones and in TRPV1-expressing NIH3T3 cells. Further, Rho-associated kinase (ROCK) phosphorylation is elevated in growth cones and neurites on micropatterned surfaces. Inhibition of RhoA/ROCK by pharmacological compounds or reduced expression of either ROCKI or ROCKII isoforms by RNAi abolishes neurite and cell alignment, confirming that RhoA/ROCK signaling mediates neurite and cell alignment to microtopographic features. These studies demonstrate that microtopographical cues recruit TRPV1 channels and downstream signaling pathways, including RhoA and ROCK, to direct neurite and cell growth. PMID:25890710

  11. Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction

    SciTech Connect

    Wang, Yingchun; Yang, Feng; Fu, Yi; Huang, Xiahe; Wang, Wei; Jiang, Xining; Gritsenko, Marina A.; Zhao, Rui; Monroe, Matthew E.; Pertz, Olivier C.; Purvine, Samuel O.; Orton, Daniel J.; Jacobs, Jon M.; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

    2011-05-20

    Abstract - Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.

  12. Nerve growth factor-induced migration of endothelial cells.

    PubMed

    Dollé, Jean-Pierre; Rezvan, Amir; Allen, Fred D; Lazarovici, Philip; Lelkes, Peter I

    2005-12-01

    Nerve growth factor (NGF) is a well known neurotropic and neurotrophic agonist in the nervous system, which recently was shown to also induce angiogenic effects in endothelial cells (ECs). To measure NGF effects on the migration of cultured ECs, an important step in neoangiogenesis, we optimized an omnidirectional migration assay using human aortic endothelial cells (HAECs) and validated the assay with human recombinant basic fibroblast growth factor (rhbFGF) and human recombinant vascular endothelial growth factor (rhVEGF). The potencies of nerve growth factor purified from various species (viper, mouse, and recombinant human) to stimulate HAEC migration was similar to that of VEGF and basic fibroblast growth factor (bFGF) (EC50 of approximately 0.5 ng/ml). Recombinant human bFGF was significantly more efficacious than either viper NGF or rhVEGF, both of which stimulated HAEC migration by approximately 30% over basal spontaneous migration. NGF-mediated stimulation of HAEC migration was completely blocked by the NGF/TrkA receptor antagonist K252a [(8R*,9S*,11S*)-(/)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,-8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(c,d,e)trindene-1-one] (30 nM) but not by the VEGF/Flk receptor antagonist SU-5416 [3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one] (250 nM), indicating a direct effect of NGF via TrkA receptor activation on HAEC migration. Viper NGF stimulation of HAEC migration was additively increased by either rhVEGF or rhbFGF, suggesting a potentiating interaction between their tyrosine kinase receptor signaling pathways. Viper NGF represents a novel pharmacological tool to investigate possible TrkA receptor subtypes in endothelial cells. The ability of NGF to stimulate migration of HAEC cells in vitro implies that this factor may play an important role in the cardiovascular system besides its well known effects in the nervous system. PMID:16123305

  13. Connective tissue growth factor induces cardiac hypertrophy through Akt signaling

    SciTech Connect

    Hayata, Nozomi; Fujio, Yasushi; Yamamoto, Yasuhiro; Iwakura, Tomohiko; Obana, Masanori; Takai, Mika; Mohri, Tomomi; Nonen, Shinpei; Maeda, Makiko; Azuma, Junichi

    2008-05-30

    In the process of cardiac remodeling, connective tissue growth factor (CTGF/CCN2) is secreted from cardiac myocytes. Though CTGF is well known to promote fibroblast proliferation, its pathophysiological effects in cardiac myocytes remain to be elucidated. In this study, we examined the biological effects of CTGF in rat neonatal cardiomyocytes. Cardiac myocytes stimulated with full length CTGF and its C-terminal region peptide showed the increase in cell surface area. Similar to hypertrophic ligands for G-protein coupled receptors, such as endothelin-1, CTGF activated amino acid uptake; however, CTGF-induced hypertrophy is not associated with the increased expression of skeletal actin or BNP, analyzed by Northern-blotting. CTGF treatment activated ERK1/2, p38 MAPK, JNK and Akt. The inhibition of Akt by transducing dominant-negative Akt abrogated CTGF-mediated increase in cell size, while the inhibition of MAP kinases did not affect the cardiac hypertrophy. These findings indicate that CTGF is a novel hypertrophic factor in cardiac myocytes.

  14. Epidermal Growth Factor-induced Vacuolar (H+)-ATPase Assembly

    PubMed Central

    Xu, Yanqing; Parmar, Amanda; Roux, Emmanuelle; Balbis, Alejandro; Dumas, Victor; Chevalier, Stephanie; Posner, Barry I.

    2012-01-01

    Using proteomics and immunofluorescence, we demonstrated epidermal growth factor (EGF) induced recruitment of extrinsic V1 subunits of the vacuolar (H+)-ATPase (V-ATPase) to rat liver endosomes. This was accompanied by reduced vacuolar pH. Bafilomycin, an inhibitor of V-ATPase, inhibited EGF-stimulated DNA synthesis and mammalian target of rapamycin complex 1 (mTORC1) activation as indicated by a decrease in eukaryotic initiation factor 4E-binding 1 (4E-BP1) phosphorylation and p70 ribosomal S6 protein kinase (p70S6K) phosphorylation and kinase activity. There was no corresponding inhibition of EGF-induced Akt and extracellular signal-regulated kinase (Erk) activation. Chloroquine, a neutralizer of vacuolar pH, mimicked bafilomycin effects. Bafilomycin did not inhibit the association of mTORC1 with Raptor nor did it affect AMP-activated protein kinase activity. Rather, the intracellular concentrations of essential but not non-essential amino acids were decreased by bafilomycin in EGF-treated primary rat hepatocytes. Cycloheximide, a translation elongation inhibitor known to augment intracellular amino acid levels, prevented the effect of bafilomycin on amino acids levels and completely reversed its inhibition of EGF-induced mTORC1 activation. In vivo administration of EGF stimulated the recruitment of Ras homologue enriched in brain (Rheb) but not mammalian target of rapamycin (mTOR) to endosomes and lysosomes. This was inhibited by chloroquine treatment. Our results suggest a role for vacuolar acidification in EGF signaling to mTORC1. PMID:22689575

  15. Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor

    PubMed Central

    Burstein, David E.; Greene, Lloyd A.

    1978-01-01

    Studies on the mechanism of action of nerve growth factor (NGF) were carried out with PC12 rat pheochromocytoma cells. PC12 cells are uniquely useful for such studies because they respond to, but (unlike normal neurons) do not require, NGF and may undergo either generation or regeneration of neurites in response to NGF. Regeneration is defined here as NGF-dependent regrowth of neurites within 24 hr after subculture of NGF-treated PC12 cells. As in cultures of normal NGF-responsive neurons, neurite regeneration by PC12 cells occurs even in the presence of high concentrations of RNA synthesis inhibitors. Generation of neurites is defined as the de novo initiation of outgrowth when PC12 cells are exposed to NGF for the first time. In contrast to regeneration, neurite generation takes place with a lag of at least 24 hr and is blocked by low concentrations of RNA synthesis inhibitors. Such findings suggest that there are both RNA synthesis-dependent and -independent pathways in the mechanism whereby NGF stimulates neurite outgrowth. In addition, NGF-treated PC12 cells undergo a time-dependent loss of the capacity for neurite regeneration after pretreatment with RNA synthesis inhibitors or withdrawal of NGF. Such findings suggest that (i) initiation of neurite outgrowth requires NGF-stimulated, RNA synthesis-dependent accumulation of intracellular material(s), (ii) once such accumulation occurs, RNA synthesis-independent regeneration can occur (but only in the presence of NGF), and (iii) the turnover of such material(s) in the absence of their replacement leads to loss of the capacity for regeneration. A tentative sequence is presented for the events whereby NGF may stimulate neurite outgrowth. PMID:310552

  16. Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds

    SciTech Connect

    Chen, Chih-Hao; Kuo, Shyh Ming; Liu, Guei-Sheung; Chen, Wan-Nan U.; Chuang, Chin-Wen; Liu, Li-Feng

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. Black-Right-Pointing-Pointer Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. Black-Right-Pointing-Pointer 3-D collagen culture of NCSCs shows an advance differentiation than 2-D culture. -- Abstract: Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 {mu}m porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

  17. Identification of a Peripheral Nerve Neurite Growth-Promoting Activity by Development and Use of an in vitro Bioassay

    NASA Astrophysics Data System (ADS)

    Sandrock, Alfred W.; Matthew, William D.

    1987-10-01

    The effective regeneration of severed neuronal axons in the peripheral nerves of adult mammals may be explained by the presence of molecules in situ that promote the effective elongation of neurites. The absence of such molecules in the central nervous system of these animals may underlie the relative inability of axons to regenerate in this tissue after injury. In an effort to identify neurite growth-promoting molecules in tissues that support effective axonal regeneration, we have developed an in vitro bioassay that is sensitive to substrate-bound factors of peripheral nerve that influence the growth of neurites. In this assay, neonatal rat superior cervical ganglion explants are placed on longitudinal cryostat sections of fresh-frozen sciatic nerve, and the regrowing axons are visualized by catecholamine histofluorescence. Axons are found to regenerate effectively over sciatic nerve tissue sections. When ganglia are similarly explanted onto cryostat sections of adult rat central nervous system tissue, however, axonal regeneration is virtually absent. We have begun to identify the molecules in peripheral nerve that promote effective axonal regeneration by examining the effect of antibodies that interfere with the activity of previously described neurite growth-promoting factors. Axonal elongation over sciatic nerve tissue was found to be sensitive to the inhibitory effects of INO (for inhibitor of neurite outgrowth), a monoclonal antibody that recognizes and inhibits a neurite growth-promoting activity from PC-12 cell-conditioned medium. The INO antigen appears to be a molecular complex of laminin and heparan sulfate proteoglycan. In contrast, a rabbit antiserum that recognizes laminin purified from mouse Engelbreth-Holm-Swarm (EHS) sarcoma, stains the Schwann cell basal lamina of peripheral nerve, and inhibits neurite growth over purified laminin substrata has no detectable effect on the rate of axonal regeneration in our assay.

  18. 5-Hydroxytryptamine 1A and 2B serotonin receptors in neurite outgrowth: involvement of early growth response protein 1.

    PubMed

    Anelli, Tonino; Cardarelli, Silvia; Ori, Michela; Nardi, Irma; Biagioni, Stefano; Poiana, Giancarlo

    2013-01-01

    Neurotransmitters play important roles in neurogenesis; in particular, acetylcholine and serotonin may regulate neurite elongation. Acetylcholine may also activate transcription factors such as early growth response protein 1 (EGR-1), which plays a role in neurite extension. N18TG2 neuroblastoma cells (which do not produce neurotransmitters and constitutively express muscarinic acetylcholine receptors) were transfected with constructs containing the cDNA for choline acetyltransferase, 5-hydroxytryptamine 1A (5-HT1A) and 5-HT2B serotonin receptors to study acetylcholine and serotonin interplay in neurite outgrowth. 5-HT1A receptor stimulation causes a decrease in EGR-1 levels and inhibition of neurite outgrowth; 5-HT2B stimulation, however, has no effect. Muscarinic cholinergic stimulation, on the other end, increases EGR-1 levels and fiber outgrowth. Inhibition of EGR-1 binding reduces fiber outgrowth activity. When both cholinergic and 5-HT1A receptors are stimulated, fiber outgrowth is restored; therefore, acetylcholine counterbalances the inhibitory effect of serotonin on neurite outgrowth. These results suggest that EGR-1 plays a role in the interplay of acetylcholine and serotonin in the regulation of neurite extension during development. PMID:24158140

  19. Channeling of developing rat corticospinal tract axons by myelin-associated neurite growth inhibitors

    SciTech Connect

    Schwab, M.E.; Schnell, L. )

    1991-03-01

    CNS myelin contains 2 membrane proteins that are potent inhibitors of neurite growth (NI-35 and NI-250). Because myelin formation starts at different times in different regions and tracts of the CNS, this inhibitory property of myelin could serve boundary and guidance functions for late-growing fiber tracts. In the rat, the corticospinal tract (CST) grows into and down the spinal cord during the first 10 postnatal days, in close proximity to the sensory tracts fasciculus cuneatus and gracilis. Immunofluorescence for myelin constituents showed that, in the rostral half of the spinal cord, the myelinating tissue of these ascending tracts surrounds the growing, myelin-free CST in a channellike fashion. Elimination of oligodendrocytes by x-irradiation of the newborn rats, or application of antibody IN-1, which neutralizes the inhibitory substrate property of CNS myelin, resulted in significant anatomical aberration of CST fibers. In particular, the tract was larger in cross-section, and aberrant CST fibers and fascicles intermixed with the neighboring sensory ascending tracts. These results assign an important channeling and guard-rail function to the oligodendrocyte-associated neurite growth inhibitors for the developing CST in the rat spinal cord.

  20. Imaging growth of neurites in conditioned hydrogel by coherent anti-stokes raman scattering microscopy.

    PubMed

    Conovaloff, Aaron; Wang, Han-Wei; Cheng, Ji-Xin; Panitch, Alyssa

    2009-10-01

    Cultured DRGs in different gel scaffolds were analyzed using CA RS microscopy to determine its possible use as a label-free imaging option for tracking cellular growth in a gel scaffold. This study demonstrates for the first time the applicability of CA RS microscopy to the imaging of live neuronal cells in GAG hydrogels. By tuning the laser beating frequency, omega(p)-omega(s), to match the vibration of C-H bonds in the cell membrane, the CA RS signal yields detailed, high-quality images of neurites with single membrane detection sensitivity. The results demonstrate that CA RS imaging allows monitoring of cellular growth in a tissue scaffold over time, with a contrast that shows comparable cellular structures to those obtained using standard fluorescent staining techniques. These findings show the potential of CARS microscopy to assist in the understanding of organogenesis processes in a tissue scaffold. PMID:20539743

  1. Antibodies directed to Neisseria gonorrhoeae impair nerve growth factor-dependent neurite outgrowth in Rat PC12 cells.

    PubMed

    Reuss, B

    2014-03-01

    In children born from mothers with prenatal infections with the Gram-negative bacterium Neisseria gonorrhoeae, schizophrenia risk is increased in later life. Since cortical neuropil formation is frequently impaired during this disease, actions of a rabbit polyclonal antiserum directed to N. gonorrhoeae on neurite outgrowth in nerve growth factor-stimulated PC12 cells were investigated here. It turned out that 10 μg/ml of the antiserum leads indeed to a significant reduction in neurite outgrowth, whereas an antiserum directed to Neisseria meningitidis had no such effect. Furthermore, reduction in neurite outgrowth could be reversed by the neuroleptic drugs haloperidol, clozapine, risperidone, and olanzapine. On the molecular level, the observed effects seem to include the known neuritogenic transcription factors FoxO3a and Stat3, since reduced neurite outgrowth caused by the antiserum was accompanied by a reduced phosphorylation of both factors. In contrast, restitution of neurite outgrowth by neuroleptic drugs revealed no correlation to the phosphorylation state of these factors. The present report gives a first hint that bacterial infections could indeed lead to impaired neuropil formation in vitro; however, the in vivo relevance of this finding for schizophrenia pathogenesis remains to be clarified in the future. PMID:24203572

  2. Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

    SciTech Connect

    Montesano, Roberto Sarkoezi, Rita; Schramek, Herbert

    2008-09-12

    Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hitherto unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.

  3. In vitro growth factor-induced bio engineering of mature articular cartilage

    PubMed Central

    Khan, Ilyas M.; Francis, Lewis; Theobald, Peter S.; Perni, Stefano; Young, Robert D.; Prokopovich, Polina; Conlan, R. Steven; Archer, Charles W.

    2013-01-01

    Articular cartilage maturation is the postnatal development process that adapts joint surfaces to their site-specific biomechanical demands. Maturation involves gross morphological changes that occur through a process of synchronised growth and resorption of cartilage and generally ends at sexual maturity. The inability to induce maturation in biomaterial constructs designed for cartilage repair has been cited as a major cause for their failure in producing persistent cell-based repair of joint lesions. The combination of growth factors FGF2 and TGFβ1 induces accelerated articular cartilage maturation in vitro such that many molecular and morphological characteristics of tissue maturation are observable. We hypothesised that experimental growth factor-induced maturation of immature cartilage would result in a biophysical and biochemical composition consistent with a mature phenotype. Using native immature and mature cartilage as reference, we observed that growth factor-treated immature cartilages displayed increased nano-compressive stiffness, decreased surface adhesion, decreased water content, increased collagen content and smoother surfaces, correlating with a convergence to the mature cartilage phenotype. Furthermore, increased gene expression of surface structural protein collagen type I in growth factor-treated explants compared to reference cartilages demonstrates that they are still in the dynamic phase of the postnatal developmental transition. These data provide a basis for understanding the regulation of postnatal maturation of articular cartilage and the application of growth factor-induced maturation in vitro and in vivo in order to repair and regenerate cartilage defects. PMID:23182922

  4. New potent accelerator of neurite outgrowth from Lawsonia inermis flower under non-fasting condition.

    PubMed

    Oda, Yoshimi; Nakashima, Souichi; Nakamura, Seikou; Yano, Mamiko; Akiyama, Masanori; Imai, Kayo; Kimura, Tomohito; Nakata, Akiko; Tani, Miyuki; Matsuda, Hisashi

    2016-07-01

    The methanolic extract of Lawsonia inermis L. (henna) showed accelerative effects on nerve growth factor-induced neurite outgrowth in PC12 cells under non-fasting conditions. To elucidate the active constituents responsible for the neuronal differentiation, we conducted a search of the constituents and examined their accelerative effects on neurite outgrowth in PC12 cells. We isolated a new acetophenone glycoside, inermioside A, which exerted a significant accelerative effect on neurite outgrowth. We also confirmed the activities of nine known compounds, including quercetin and lalioside. In addition, we found that quercetin, one of the active constituents, increased Vav3 mRNA expression. PMID:26936787

  5. Microfluidic Generation of Haptotactic Gradients through 3D Collagen Gels for Enhanced Neurite Growth

    PubMed Central

    Sundararaghavan, Harini G.; Masand, Shirley N.

    2011-01-01

    Abstract We adapted a microfluidic system used previously to generate durotactic gradients of stiffness in a 3D collagen gel, to produce haptotactic gradients of adhesive ligands through the collagen gel. Oligopeptide sequences that included bioactive peptide sequences from laminin, YIGSR, or IKVAV, were grafted separately onto type I collagen using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Solutions of peptide-grafted collagen and untreated collagen were then used as source and sink input solutions, respectively, in an H-shaped microfluidic network fabricated using traditional soft lithography. One-dimensional gradients of the peptide-grafted collagen solution were generated in the channel that connected the source and sink channels, and these gradients became immobilized upon self-assembly of the collagen into a 3D fibrillar gel. The slope and average concentration of the gradients were adjusted by changing the concentration of the source solutions and by changing the length of the cross-channel. A separate, underlying channel in the microfluidic construct allowed the introduction of a chick embryo dorsal root ganglion into the network. Neurites from these explants grew significantly longer up steep gradients of YIGSR, but shallow gradients of IKVAV in comparison to untreated collagen controls. When these two gradients were presented in combination, the bias in growth acceleration was the largest and most consistent. No differences were observed in the number of neurites choosing to grow up or down the gradients in any condition. These results suggest that the incorporation of distinct gradients of multiple bioactive ligands can improve directional acceleration of regenerating axons. PMID:21473683

  6. Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth.

    PubMed

    Arastoo, Mohammed; Hacker, Christian; Popovics, Petra; Lucocq, John M; Stewart, Alan J

    2016-02-01

    Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCη2 by comparing immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling for PLCη2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCη2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of the PLCη2 EF-hand domain and articulation of PLCη2 with LIMK-1 in regulating neuritogenesis. PMID:26671787

  7. TTLL7 Is a Mammalian β-Tubulin Polyglutamylase Required for Growth of MAP2-positive Neurites*S

    PubMed Central

    Ikegami, Koji; Mukai, Masahiro; Tsuchida, Jun-ichi; Heier, Robb L.; MacGregor, Grant R.; Setou, Mitsutoshi

    2011-01-01

    Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform β-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for β-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to β-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for β-tubulin and not for α-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated β-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of β-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (microtubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated β-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a β-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells. PMID:16901895

  8. Antibody-mediated inhibition of Nogo-A signaling promotes neurite growth in PC-12 cells

    PubMed Central

    Yazdi, Iman K; Taghipour, Nima; Hmaidan, Sarah; Palomba, Roberto; Scaria, Shilpa; Munoz, Alvaro; Boone, Timothy B; Tasciotti, Ennio

    2016-01-01

    The use of a monoclonal antibody to block the neurite outgrowth inhibitor Nogo-A has been of great interest for promoting axonal recovery as a treatment for spinal cord injury. While several cellular and non-cellular assays have been developed to quantify the bioactive effects of Nogo-A signaling, demand still exists for the development of a reliable approach to characterize the effectiveness of the anti-Nogo-A antibody. In this study, we developed and validated a novel cell-based approach to facilitate the biological quantification of a Nogo-A antibody using PC-12 cells as an in vitro neuronal cell model. Changes in the mRNA levels of the neuronal differentiation markers, growth-associated protein 43 and neurofilament light-polypeptide, suggest that activation of the Nogo-A pathway suppresses axonal growth and dendrite formation in the tested cell line. We found that application of anti-Nogo-A monoclonal antibody can significantly enhance the neuronal maturity of PC-12 cells by blocking the Nogo-A inhibitory effects, providing enhanced effects on neural maturity at the molecular level. No adverse effects were observed on cell viability. PMID:27027860

  9. Expression of Wnt Receptors in Adult Spiral Ganglion Neurons: Frizzled 9 Localization at Growth Cones of Regenerating Neurites

    PubMed Central

    Shah, S. M.; Kang, Y.-J.; Christensen, B. L.; Feng, A. S.; Kollmar, R.

    2009-01-01

    Little is known about signaling pathways, besides those of neurotrophic factors, that are operational in adult spiral ganglion neurons. In patients with sensorineural hearing loss, such pathways could eventually be targeted to stimulate and guide neurite outgrowth from the remnants of the spiral ganglion towards a cochlear implant, thereby improving the fidelity of sound transmission. To systematically identify neuronal receptors for guidance cues in the adult cochlea, we conducted a genome-wide cDNA microarray screen with two-month-old CBA/CaJ mice. A meta-analysis of our data and those from older mice in two other studies revealed the presence of neuronal transmembrane receptors that represent all four established guidance pathways—ephrin, netrin, semaphorin, and slit—in the mature cochlea as late as 15 months. In addition, we observed the expression of all known receptors for the Wnt morphogens, whose neuronal guidance function has only recently been recognized. In situ hybridizations located the mRNAs of the Wnt receptors frizzled 1, 4, 6, 9, and 10 specifically in adult spiral ganglion neurons. Finally, frizzled 9 protein was found in the growth cones of adult spiral ganglion neurons that were regenerating neurites in culture. We conclude from our results that adult spiral ganglion neurons are poised to respond to neurite damage, owing to the constitutive expression of a large and diverse collection of guidance receptors. Wnt signaling, in particular, emerges as a candidate pathway for guiding neurite outgrowth towards a cochlear implant after sensorineural hearing loss. PMID:19716861

  10. Synthesis of poly(ester-carbonate) with a pendant acetylcholine analog for promoting neurite growth.

    PubMed

    Xing, Dongming; Ma, Lie; Gao, Changyou

    2014-10-01

    The modification of biodegradable polyesters with bioactive molecules has become an important strategy for controlling neuron adhesion and neurite outgrowth in nerve regeneration. In this study we report a biodegradable poly(ester-carbonate) with a pendant acetylcholine analog, which a neurotransmitter for the enhancement of neuron adhesion and outgrowth. The acetylcholine-functionalized poly(ester-carbonate) (Ach-P(LA-ClTMC)) was prepared by copolymerizing l-lactide (LA) and 5-methyl-5-chloroethoxycarbonyl trimethylene carbonate (ClTMC), followed by quaternization with trimethylamine. The acetylcholine analog content could be modulated by changing the molar feeding fraction of ClTMC. The incorporation of the acetylcholine analog improved the hydrophilicity of the films, but the acetylcholine analog content did not significantly influence the surface morphology of the acetylcholine-functionalized films. The results of PC12 cell culture showed that the acetylcholine analog promoted cell viability and neurite outgrowth in a concentration-dependent manner. The longest length of neurite and the percentage of cells bearing neurites were obtained on the Ach-P(LA-ClTMC)-10 film. All the results indicate that the integration of the acetylcholine analog at an appropriate fraction could be an effective strategy for optimizing the existing biodegradable polyesters for nerve regeneration applications. PMID:24998182

  11. Mo polyoxometalate nanoparticles inhibit tumor growth and vascular endothelial growth factor induced angiogenesis

    NASA Astrophysics Data System (ADS)

    Zheng, Wenjing; Yang, Licong; Liu, Ying; Qin, Xiuying; Zhou, Yanhui; Zhou, Yunshan; Liu, Jie

    2014-06-01

    Tumor growth depends on angiogenesis, which can furnish the oxygen and nutrients that proliferate tumor cells. Thus, blocking angiogenesis can be an effective strategy to inhibit tumor growth. In this work, three typical nanoparticles based on polyoxometalates (POMs) have been prepared; we investigated their capability as antitumor and anti-angiogenesis agents. We found that Mo POM nanoparticles, especially complex 3, inhibited the growth of human hepatocellular liver carcinoma cells (HepG2) through cellular reactive oxygen species levels’ elevation and mitochondrial membrane potential damage. Complex 3 also suppressed the proliferation, migration, and tube formation of endothelial cells in vitro and chicken chorioallantoic membrane development ex vivo. Furthermore, western blot analysis of cell signaling molecules indicated that Mo POMs blocked the vascular endothelial growth factor receptor 2-mediated ERK1/2 and AKT signaling pathways in endothelial cells. Using transmission electron microscopy, we demonstrated their cellular uptake and localization within the cytoplasm of HepG2 cells. These results indicate that, owing to the extraordinary physical and chemical properties, Mo POM nanoparticles can significantly inhibit tumor growth and angiogenesis, which makes them potential drug candidates in anticancer and anti-angiogenesis therapies.

  12. Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

    PubMed Central

    O'Driscoll, K R; Teng, K K; Fabbro, D; Greene, L A; Weinstein, I B

    1995-01-01

    The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells. Images PMID:7626808

  13. Control of Retinal Ganglion Cell Positioning and Neurite Growth: Combining 3D Printing with Radial Electrospun Scaffolds.

    PubMed

    Kador, Karl E; Grogan, Shawn P; Dorthé, Erik W; Venugopalan, Praseeda; Malek, Monisha F; Goldberg, Jeffrey L; D'lima, Darryl D

    2016-02-01

    Retinal ganglion cells (RGCs) are responsible for the transfer of signals from the retina to the brain. As part of the central nervous system, RGCs are unable to regenerate following injury, and implanted cells have limited capacity to orient and integrate in vivo. During development, secreted guidance molecules along with signals from extracellular matrix and the vasculature guide cell positioning, for example, around the fovea, and axon outgrowth; however, these changes are temporally regulated and are not the same in the adult. Here, we combine electrospun cell transplantation scaffolds capable of RGC neurite guidance with thermal inkjet 3D cell printing techniques capable of precise positioning of RGCs on the scaffold surface. Optimal printing parameters are developed for viability, electrophysiological function and, neurite pathfinding. Different media, commonly used to promote RGC survival and growth, were tested under varying conditions. When printed in growth media containing both brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), RGCs maintained survival and normal electrophysiological function, and displayed radial axon outgrowth when printed onto electrospun scaffolds. These results demonstrate that 3D printing technology may be combined with complex electrospun surfaces in the design of future retinal models or therapies. PMID:26729061

  14. Expression and function of fibroblast growth factor-inducible 14 in human corneal myofibroblasts.

    PubMed

    Ebihara, Nobuyuki; Nakayama, Masafumi; Tokura, Tomoko; Ushio, Hiroko; Murakami, Akira

    2009-08-01

    The interaction of fibroblast growth factor-inducible 14 (Fn14) and, its ligand tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is known to be important in wound healing of tissues. However, to our knowledge, expression and function of Fn14 in corneal myofibroblasts, which have a crucial role in wound healing of corneal stroma, has not been investigated. In this study, we investigated the expression and function of Fn14 in corneal myofibroblasts. Expression of Fn14 protein was assessed by flow cytometry. Corneal myofibroblasts showed strong expression of Fn14 protein, while keratocytes did not. TGF-beta(1) promoted the differentiation of keratocytes into corneal myofibroblasts, and induced Fn14 expression. These data reveal that keratocytes phenotype determines the level of Fn14 expression. ELISA was used to detect chemokines and matrix metalloproteinases in the supernatant of corneal myofibroblasts cultured with or without stimulation by TWEAK and/or TGF-beta(1). TWEAK increased the production of IL-8, MCP-1, and RANTES by corneal myofibroblasts via Fn14. TGF-beta(1) augmented the TWEAK-induced production of these chemokines. TWEAK also increased the production of MMP-1 and -3 by corneal myofibroblasts via Fn14, while TGF-beta(1) inhibited this effect of TWEAK on MMP production. TWEAK-induced phosphorylation of NF-kappaB and MAP kinase in corneal myofibroblasts. Furthermore, TWEAK partially inhibited the differentiation of keratocytes into corneal myofibroblasts promoted by TGF-beta(1). These data suggest that the Fn14/TWEAK system may have several roles in wound healing by corneal myofibroblasts. In the future, modulation of the TWEAK/Fn14 system may become a novel approach for control corneal wound healing. PMID:19344712

  15. Nerve Growth Factor Secretion From Pulp Fibroblasts is Modulated by Complement C5a Receptor and Implied in Neurite Outgrowth.

    PubMed

    Chmilewsky, Fanny; Ayaz, Warda; Appiah, James; About, Imad; Chung, Seung-Hyuk

    2016-01-01

    Given the importance of sensory innervation in tooth vitality, the identification of signals that control nerve regeneration and the cellular events they induce is essential. Previous studies demonstrated that the complement system, a major component of innate immunity and inflammation, is activated at the injured site of human carious teeth and plays an important role in dental-pulp regeneration via interaction of the active Complement C5a fragment with pulp progenitor cells. In this study, we further determined the role of the active fragment complement C5a receptor (C5aR) in dental nerve regeneration in regards to local secretion of nerve growth factor (NGF) upon carious injury. Using ELISA and AXIS co-culture systems, we demonstrate that C5aR is critically implicated in the modulation of NGF secretion by LTA-stimulated pulp fibroblasts. The NGF secretion by LTA-stimulated pulp fibroblasts, which is negatively regulated by C5aR activation, has a role in the control of the neurite outgrowth length in our axon regeneration analysis. Our data provide a scientific step forward that can guide development of future therapeutic tools for innovative and incipient interventions targeting the dentin-pulp regeneration process by linking the neurite outgrowth to human pulp fibroblast through complement system activation. PMID:27539194

  16. Mathematical Relationships between Neuron Morphology and Neurite Growth Dynamics in Drosophila melanogaster Larva Class IV Sensory Neurons

    NASA Astrophysics Data System (ADS)

    Ganguly, Sujoy; Liang, Xin; Grace, Michael; Lee, Daniel; Howard, Jonathon

    The morphology of neurons is diverse and reflects the diversity of neuronal functions, yet the principles that govern neuronal morphogenesis are unclear. In an effort to better understand neuronal morphogenesis we will be focusing on the development of the dendrites of class IV sensory neuron in Drosophila melanogaster. In particular we attempt to determine how the the total length, and the number of branches of dendrites are mathematically related to the dynamics of neurite growth and branching. By imaging class IV neurons during early embryogenesis we are able to measure the change in neurite length l (t) as a function of time v (t) = dl / dt . We found that the distribution of v (t) is well characterized by a hyperbolic secant distribution, and that the addition of new branches per unit time is well described by a Poisson process. Combining these measurements with the assumption that branching occurs with equal probability anywhere along the dendrite we were able to construct a mathematical model that provides reasonable agreement with the observed number of branches, and total length of the dendrites of the class IV sensory neuron.

  17. Nerve Growth Factor Secretion From Pulp Fibroblasts is Modulated by Complement C5a Receptor and Implied in Neurite Outgrowth

    PubMed Central

    Chmilewsky, Fanny; Ayaz, Warda; Appiah, James; About, Imad; Chung, Seung-Hyuk

    2016-01-01

    Given the importance of sensory innervation in tooth vitality, the identification of signals that control nerve regeneration and the cellular events they induce is essential. Previous studies demonstrated that the complement system, a major component of innate immunity and inflammation, is activated at the injured site of human carious teeth and plays an important role in dental-pulp regeneration via interaction of the active Complement C5a fragment with pulp progenitor cells. In this study, we further determined the role of the active fragment complement C5a receptor (C5aR) in dental nerve regeneration in regards to local secretion of nerve growth factor (NGF) upon carious injury. Using ELISA and AXIS co-culture systems, we demonstrate that C5aR is critically implicated in the modulation of NGF secretion by LTA-stimulated pulp fibroblasts. The NGF secretion by LTA-stimulated pulp fibroblasts, which is negatively regulated by C5aR activation, has a role in the control of the neurite outgrowth length in our axon regeneration analysis. Our data provide a scientific step forward that can guide development of future therapeutic tools for innovative and incipient interventions targeting the dentin-pulp regeneration process by linking the neurite outgrowth to human pulp fibroblast through complement system activation. PMID:27539194

  18. Quercetin 3-O-glucoside suppresses epidermal growth factor-induced migration by inhibiting EGFR signaling in pancreatic cancer cells.

    PubMed

    Lee, Jungwhoi; Han, Song-I; Yun, Jeong-Hun; Kim, Jae Hoon

    2015-12-01

    Pancreatic cancer is one of the most dangerous cancers and is associated with a grave prognosis. Despite increased knowledge of the complex signaling networks responsible for progression of pancreatic cancer, many challenging therapies have fallen short of expectations. In this study, we examined the anti-migratory effect of quercetin 3-O-glucoside in epidermal growth factor-induced cell migration by inhibiting EGF receptor (EGFR) signaling in several human pancreatic cancer cell lines. Treatment with quercetin, quercetin 3-O-glucoside, and quercetin 7-O-glucoside differentially suppressed epidermal growth factor-induced migration activity of human pancreatic cancer cells. In particular, quercetin 3-O-glucoside strongly inhibited the infiltration activity of pancreatic cancer cells in a dose-dependent manner. Furthermore, quercetin 3-O-glucoside exerted the anti-migratory effect even at a relatively low dose compared with other forms of quercetin. The anti-tumor effects of quercetin 3-O-glucoside were mediated by selectively inhibiting the EGFR-mediated FAK, AKT, MEK1/2, and ERK1/2 signaling pathway. Combinatorial treatment with quercetin 3-O-glucoside plus gemcitabine showed the synergistic anti-migratory effect on epidermal growth factor-induced cell migration in human pancreatic cancer cell lines. These results suggest that quercetin 3-O-glucoside has potential for anti-metastatic therapy in human pancreatic cancer. PMID:26109002

  19. Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons

    PubMed Central

    Cohen, Matthew R.; Johnson, William M.; Pilat, Jennifer M.; Kiselar, Janna; DeFrancesco-Lisowitz, Alicia; Zigmond, Richard E.

    2015-01-01

    Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca2+-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca2+-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca2+ signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca2+ signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function. PMID:26416880

  20. Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons.

    PubMed

    Cohen, Matthew R; Johnson, William M; Pilat, Jennifer M; Kiselar, Janna; DeFrancesco-Lisowitz, Alicia; Zigmond, Richard E; Moiseenkova-Bell, Vera Y

    2015-12-01

    Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca(2+)-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca(2+)-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca(2+) signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca(2+) signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function. PMID:26416880

  1. Photopolymerized microfeatures for directed spiral ganglion neurite and Schwann cell growth

    PubMed Central

    Tuft, Bradley W.; Li, Shufeng; Xu, Linjing; Clarke, Joseph C.; White, Scott P.; Guymon, Bradley A.; Perez, Krystian X.; Hansen, Marlan R.; Guymon, C. Allan

    2015-01-01

    Cochlear implants (CIs) provide auditory perception to individuals with severe hearing impairment. However, their ability to encode complex auditory stimuli is limited due, in part, to poor spatial resolution caused by electrical current spread in the inner ear. Directing nerve cell processes towards target electrodes may reduce the problematic current spread and improve stimulatory specificity. In this work, photopolymerization was used to fabricate micro- and nano-patterned methacrylate polymers to probe the extent of spiral ganglion neuron (SGN) neurite and Schwann cell (SGSC) contact guidance based on variations in substrate topographical cues. Micropatterned substrates are formed in a rapid, single-step reaction by selectively blocking light with photomasks which have parallel line-space gratings with periodicities of 10 – 100 μm. Channel amplitudes of 250 nm – 10 μm are generated by modulating UV exposure time, light intensity, and photoinitiator concentration. Gradual transitions are observed between ridges and grooves using scanning electron and atomic force microscopy. The transitions stand in contrast to vertical features generated via etching lithographic techniques. Alignment of neural elements increases significantly with increasing feature amplitude and constant periodicity, as well as with decreasing periodicity and constant amplitude. SGN neurite alignment strongly correlates (r = 0.93) with maximum feature slope. Multiple neuronal and glial types orient to the patterns with varying degrees of alignment. This work presents a method to fabricate gradually-sloping micropatterns for cellular contact guidance studies and demonstrates spatial control of inner ear neural elements in response to micro- and nano-scale surface topography. PMID:23069708

  2. Autophagy inhibition in endogenous and nutrient-deprived conditions reduces dorsal root ganglia neuron survival and neurite growth in vitro.

    PubMed

    Clarke, Joseph-Patrick; Mearow, Karen

    2016-07-01

    Peripheral neuropathies can result in cytoskeletal changes in axons, ultimately leading to Wallerian degeneration and cell death. Recently, autophagy has been studied as a potential target for improving axonal survival and growth during peripheral nerve damage. This study investigates the influence of autophagy on adult dorsal root ganglia (DRG) neuron survival and axonal growth under control and nutrient deprivation conditions. Constitutive autophagy was modulated with pharmacological activators (rapamycin; Rapa) and inhibitors (3-methyladenine, bafilomycin A1) in conjunction with either a nutrient-stable environment (standard culture medium) or a nutrient-deprived environment (Hank's balanced salt solution + Ca(2+) /Mg(2+) ). The results demonstrated that autophagy inhibition decreased cell viability and reduced neurite growth and branching complexity. Although autophagy was upregulated with nutrient deprivation compared with the control, it was not further activated by rapamycin, suggesting a threshold level of autophagy. Overall, both cellular and biochemical approaches combined to show the influence of autophagy on adult DRG neuron survival and growth. © 2016 Wiley Periodicals, Inc. PMID:27018986

  3. NeuronGrowth, a software for automatic quantification of neurite and filopodial dynamics from time-lapse sequences of digital images.

    PubMed

    Fanti, Zian; Martinez-Perez, M Elena; De-Miguel, Francisco F

    2011-10-01

    We developed NeuronGrowth, a software for the automatic quantification of extension and retraction of neurites and filopodia, from time-lapse sequences of two-dimensional digital micrographs. NeuronGrowth requires a semiautomatic characterization of individual neurites in a reference frame, which is then used for automatic tracking and measurement of every neurite over the whole image sequence. Modules for sequence alignment, background subtraction, flat field correction, light normalization, and cropping have been integrated to improve the quality of the analysis. Moreover, NeuronGrowth incorporates a deconvolution filter that corrects the shadow-cast effect of differential interference contrast (DIC) images. NeuronGrowth was tested by analyzing the formation of outgrowth patterns by individual leech neurons cultured under two different conditions. Phase contrast images were obtained from neurons plated on CNS homogenates and DIC images were obtained from similar neurons plated on ganglion capsules as substrates. Filopodia were measured from fluorescent growth-cones of chick dorsal root ganglion cells. Quantitative data of neurite extension and retraction obtained by three different users applying NeuronGrowth and two other manually operated software packages were similar. However, NeuronGrowth required less user participation and had a better time performance when compared with the other software packages. NeuronGrowth may be used in general to quantify the dynamics of tubular structures such as blood vessels. NeuronGrowth is a free plug-in for the free software ImageJ and can be downloaded along with a user manual, a troubleshooting section and other information required for its use from http://www.ifc.unam.mx or http://www.ifc.unam.mx/ffm/index.html. PMID:21913334

  4. Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation

    PubMed Central

    Winans, Amy M; Collins, Sean R; Meyer, Tobias

    2016-01-01

    Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon. DOI: http://dx.doi.org/10.7554/eLife.12387.001 PMID:26836307

  5. Nerve growth factor-induced differentiation of PC12 cells is accompanied by elevated adenylyl cyclase activity.

    PubMed

    Yung, H S; Lai, K H; Chow, K B S; Ip, N Y; Tsim, K W K; Wong, Y H; Wu, Z; Wise, H

    2010-01-01

    Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on G(s)-coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of Gα(s). These findings highlight that NGF has a subtle influence on adenylyl cyclase activity in PC12 cells which may influence more than the neurite extension process classically associated with neuronal differentiation. PMID:20389133

  6. Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

    SciTech Connect

    Wright, K.T.; Seabright, R.; Logan, A.; Lilly, A.J.; Khanim, F.; Bunce, C.M.; Johnson, W.E.B.

    2010-07-16

    Research highlights: {yields} Extracellular Nm23H1 stimulates nerve growth. {yields} Extracellular Nm23H1 provides pathfinding cues to growth cones. {yields} The neurotrophic activity of Nm23H1 is independent of NDP kinase activity. {yields} The neurotrophic activity of Nm23H1 is independent of NGF. -- Abstract: The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.

  7. SOXC Transcription Factors Induce Cartilage Growth Plate Formation in Mouse Embryos by Promoting Noncanonical WNT Signaling.

    PubMed

    Kato, Kenji; Bhattaram, Pallavi; Penzo-Méndez, Alfredo; Gadi, Abhilash; Lefebvre, Véronique

    2015-09-01

    Growth plates are specialized cartilage structures that ensure the elongation of most skeletal primordia during vertebrate development. They are made by chondrocytes that proliferate in longitudinal columns and then progress in a staggered manner towards prehypertrophic, hypertrophic and terminal maturation. Complex molecular networks control the formation and activity of growth plates, but remain incompletely understood. We investigated here the importance of the SoxC genes, which encode the SOX4, SOX11 and SOX12 transcription factors, in growth plates. We show that the three genes are expressed robustly in perichondrocytes and weakly in growth plate chondrocytes. SoxC(Prx1Cre) mice, which deleted SoxC genes in limb bud skeletogenic mesenchyme, were born with tiny appendicular cartilage primordia because of failure to form growth plates. In contrast, SoxC(Col2Cre) and SoxC(ATC) mice, which deleted SoxC genes primarily in chondrocytes, were born with mild dwarfism and fair growth plates. Chondrocytes in the latter mutants matured normally, but formed irregular columns, proliferated slowly and died ectopically. Asymmetric distribution of VANGL2 was defective in both SoxC(Prx1Cre) and SoxC(ATC) chondrocytes, indicating impairment of planar cell polarity, a noncanonical WNT signaling pathway that controls growth plate chondrocyte alignment, proliferation and survival. Accordingly, SoxC genes were necessary in perichondrocytes for expression of Wnt5a, which encodes a noncanonical WNT ligand required for growth plate formation, and in chondrocytes and perichondrocytes for expression of Fzd3 and Csnk1e, which encode a WNT receptor and casein kinase-1 subunit mediating planar cell polarity, respectively. Reflecting the differential strengths of the SOXC protein transactivation domains, SOX11 was more powerful than SOX4, and SOX12 interfered with the activity of SOX4 and SOX11. Altogether, these findings provide novel insights into the molecular regulation of skeletal

  8. Growth factor induced proliferation, migration, and lumen formation of rat endometrial epithelial cells in vitro.

    PubMed

    Islam, Md Rashedul; Yamagami, Kazuki; Yoshii, Yuka; Yamauchi, Nobuhiko

    2016-06-17

    Endometrial modulation is essential for the preservation of normal uterine physiology, and this modulation is driven by a number of growth factors. The present study investigated the mitogenic, motogenic, and morphogenic effects of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) on rat endometrial epithelial (REE) cells. The REE cells were isolated and cultured and then characterized based on their morphology and their expression of epithelial cell markers. The MTT assay revealed that EGF and HGF induce proliferation of REE cells. Consistent with increased proliferation, we found that the cell cycle regulatory factor Cyclin D1 was also upregulated upon EGF and HGF addition. REE cell migration was prompted by EGF, as observed with the Oris Cell Migration Assay. The morphogenic impact of growth factors on REE cells was studied in a three-dimensional BD Matrigel cell culture system, wherein these growth factors also increased the frequency of lumen formation. In summary, we show that EGF and HGF have a stimulatory effect on REE cells, promoting proliferation, cell migration, and lumen formation. Our findings provide important insights that further the understanding of endometrial regeneration and its regulation. PMID:26946922

  9. Growth factor induced proliferation, migration, and lumen formation of rat endometrial epithelial cells in vitro

    PubMed Central

    ISLAM, Md. Rashedul; YAMAGAMI, Kazuki; YOSHII, Yuka; YAMAUCHI, Nobuhiko

    2016-01-01

    Endometrial modulation is essential for the preservation of normal uterine physiology, and this modulation is driven by a number of growth factors. The present study investigated the mitogenic, motogenic, and morphogenic effects of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) on rat endometrial epithelial (REE) cells. The REE cells were isolated and cultured and then characterized based on their morphology and their expression of epithelial cell markers. The MTT assay revealed that EGF and HGF induce proliferation of REE cells. Consistent with increased proliferation, we found that the cell cycle regulatory factor Cyclin D1 was also upregulated upon EGF and HGF addition. REE cell migration was prompted by EGF, as observed with the Oris Cell Migration Assay. The morphogenic impact of growth factors on REE cells was studied in a three-dimensional BD Matrigel cell culture system, wherein these growth factors also increased the frequency of lumen formation. In summary, we show that EGF and HGF have a stimulatory effect on REE cells, promoting proliferation, cell migration, and lumen formation. Our findings provide important insights that further the understanding of endometrial regeneration and its regulation. PMID:26946922

  10. ONTOGENY OF PROTEINS ASSOCIATED WITH NEURITE GROWTH AND SYNAPTOGENESIS IN CEREBELLAR GRANULE CELLS IN VITRO.

    EPA Science Inventory

    In vitro techniques may be useful in screening for effects of developmental neurotoxicants. Previously, we characterized changes in biochemical markers associated with neuronal development in a PC12 cell model of differentiation and growth. The current research extended these stu...

  11. Expression of cyr61, a growth factor-inducible immediate-early gene.

    PubMed Central

    O'Brien, T P; Yang, G P; Sanders, L; Lau, L F

    1990-01-01

    A set of immediate-early genes that are rapidly activated by serum or purified platelet-derived growth factor in mouse 3T3 fibroblasts has been previously identified. Among these genes, several are related to known or putative transcription factors and growth factors, supporting the notion that some of these genes encode regulatory molecules important to cell growth. We show here that a member of this set of genes, cyr61 (originally identified by its cDNA 3CH61), encodes a 379-amino-acid polypeptide rich in cysteine residues. cyr61 can be induced through protein kinase C-dependent and -independent pathways. Unlike many immediate-early genes that are transiently expressed, the cyr61 mRNA is accumulated from the G0/G1 transition through mid-G1. This expression pattern is due to persistent transcription, while the mRNA is rapidly turned over during the G0/G1 transition and in mid-G1 at the same rate. In logarithmically growing cells, the cyr61 mRNA level is constant throughout the cell cycle. Cyr61 contains an N-terminal secretory signal sequence; however, it is not detected in the culture medium by immunoprecipitation. Cyr61 is synthesized maximally at 1 to 2 h after serum stimulation and has a short half-life within the cell. Images PMID:2355916

  12. Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

    SciTech Connect

    Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

    2009-04-15

    Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G{sub 1} phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

  13. ASSESSMENT OF PC12 CELL DIFFERENTIATION AND NEURITE GROWTH: A COMPARISON OF MORPHOLOGICAL AND NEUROCHEMICAL MEASURES.

    EPA Science Inventory

    In order to screen large numbers of chemicals for their potential to produce developmental neurotoxicity new, in vitro methods are needed. One approach is to develop methods based on the biologic processes which underlie brain development including the growth and maturation of ce...

  14. Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

    PubMed Central

    Housley, R M; Morris, C F; Boyle, W; Ring, B; Biltz, R; Tarpley, J E; Aukerman, S L; Devine, P L; Whitehead, R H; Pierce, G F

    1994-01-01

    Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut. Images PMID:7962522

  15. NOVEL ATYPICAL PKC INHIBITORS PREVENT VASCULAR ENDOTHELIAL GROWTH FACTOR-INDUCED BLOOD-RETINAL BARRIER DYSFUNCTION

    PubMed Central

    Titchenell, Paul M.; Lin, Cheng-Mao; Keil, Jason M.; Sundstrom, Jeffrey M.; Smith, Charles D.; Antonetti, David A.

    2013-01-01

    SYNOPSIS Pro-inflammatory cytokines and growth factors such as vascular endothelial growth factor (VEGF) contribute to the loss of the blood-retinal barrier (BRB) and subsequent macular edema in various retinal pathologies. VEGF signaling requires conventional PKC (PKCβ) activity; however, PKCβ inhibition only partially prevents VEGF-induced endothelial permeability and does not affect pro-inflammatory cytokine-induced permeability suggesting the involvement of alternative signaling pathways. Here, we provide evidence for the involvement of atypical protein kinase C (aPKC) signaling in VEGF-induced endothelial permeability and identify a novel class of inhibitors of aPKC that prevent BRB breakdown in vivo. Genetic and pharmacological manipulations of aPKC isoforms were used to assess their contribution to endothelial permeability in culture. A chemical library was screened using an in vitro kinase assay to identify novel small molecule inhibitors and further medicinal chemistry was performed to delineate a novel pharmacophore. We demonstrate that aPKC isoforms are both sufficient and required for VEGF-induced endothelial permeability. Furthermore, these specific, potent, non-competitive, small molecule inhibitors prevented VEGF-induced tight junction internalization and retinal endothelial permeability in response to VEGF in both primary culture and in rodent retina. These data suggest that aPKC inhibition with 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the BRB in retinal diseases such as diabetic retinopathy or uveitis and the blood-brain barrier (BBB) in the presence of brain tumors. PMID:22721706

  16. ENDOGLIN is dispensable for vasculogenesis, but required for vascular endothelial growth factor-induced angiogenesis.

    PubMed

    Liu, Zhen; Lebrin, Franck; Maring, Janita A; van den Driesche, Sander; van der Brink, Stieneke; van Dinther, Maarten; Thorikay, Midory; Martin, Sabrina; Kobayashi, Kazuki; Hawinkels, Lukas J A C; van Meeteren, Laurens A; Pardali, Evangelia; Korving, Jeroen; Letarte, Michelle; Arthur, Helen M; Theuer, Charles; Goumans, Marie-José; Mummery, Christine; ten Dijke, Peter

    2014-01-01

    ENDOGLIN (ENG) is a co-receptor for transforming growth factor-β (TGF-β) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis. PMID:24489709

  17. ENDOGLIN Is Dispensable for Vasculogenesis, but Required for Vascular Endothelial Growth Factor-Induced Angiogenesis

    PubMed Central

    van der Brink, Stieneke; van Dinther, Maarten; Thorikay, Midory; Martin, Sabrina; Kobayashi, Kazuki; Hawinkels, Lukas J. A. C.; van Meeteren, Laurens A.; Pardali, Evangelia; Korving, Jeroen; Letarte, Michelle; Arthur, Helen M.; Theuer, Charles; Goumans, Marie-José; Mummery, Christine; ten Dijke, Peter

    2014-01-01

    ENDOGLIN (ENG) is a co-receptor for transforming growth factor-β (TGF-β) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis. PMID:24489709

  18. Involvement of hepatocyte growth factor-induced epithelial-mesenchymal transition in human adenomyosis.

    PubMed

    Khan, Khaleque Newaz; Kitajima, Michio; Hiraki, Koichi; Fujishita, Akira; Nakashima, Masahiro; Masuzaki, Hideaki

    2015-02-01

    Adenomyosis is commonly believed to arise from the basalis endometrium. As an estromedin growth factor, hepatocyte growth factor (HGF) exhibits multiple functions in endometriosis, a disease commonly believed to arise from the functionalis endometrium. Here, we investigated the role of HGF in the occurrence of epithelial-mesenchymal transition (EMT) in adenomyosis. Full-thickness-biopsy specimens from endometrium to myometrium were collected after hysterectomy from women with and without adenomyosis. The relationship between HGF and E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell markers) was examined at the gene and protein levels using endometrial epithelial cells (EECs) in culture and tissues by quantitative RT-PCR and immunohistochemistry. The gene and protein expressions of two transcriptional repressors of E-cadherin, SLUG and SNAIL, were examined using Ishikawa cells and in response to HGF and estrogen (E2). HGF down-regulated E-cadherin and up-regulated N-cadherin mRNA expression in EECs, and an inverse relationship in protein expression between HGF and E-cadherin was observed in basalis endometria derived from women with diffuse and focal adenomyosis. HGF induced morphological changes of EECs from a cobblestone-like appearance to spindle-shaped cells and promoted migration of EECs. Ishikawa cells exhibited up-regulation of SLUG/SNAIL gene expression in response to both HGF and E2 with an additive effect between them. HGF- and E2-promoted SLUG/SNAIL gene expression was significantly abrogated after pretreatment of cells with anti-HGF antibody or ICI 182720, an estrogen receptor antagonist. HGF may be involved in gland invagination deep into the myometrium by inducing EMT at the endo-myometrial junction in women with adenomyosis. PMID:25505196

  19. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    SciTech Connect

    Nagata, Yosuke Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  20. Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization.

    PubMed Central

    Drake, C J; Little, C D

    1995-01-01

    Vascular endothelial growth factor (VEGF) is a potent and specific endothelial mitogen that is able to induce angiogenesis in vivo [Leung, D. W., Cachianes, G., Kuang, W.-J., Goeddel, D. V. & Ferrara, N. (1989) Science 246 1306-1309]. To determine if VEGF also influences the behavior of primordial endothelial cells, we used an in vivo vascular assay based on the de novo formation of vessels. Japanese quail embryos injected with nanomolar quantities of the 165-residue form of VEGF at the onset of vasculogenesis exhibited profoundly altered vessel development. In fact, the overall patterning of the vascular network was abnormal in all VEGF-injected embryos. The malformations were attributable to two specific endothelial cell activities: (i) inappropriate neovascularization in normally avascular areas and (ii) the unregulated, excessive fusion of vessels. In the first instance, supernumerary vessels directly linked the inflow channel of the heart to the aortic outflow channel. The second aberrant activity led to the formation of vessels with abnormally large lumens. Ultimately, unregulated vessel fusion generated massive vascular sacs that obliterated the identity of individual vessels. These observations show that exogenous VEGF has an impact on the behavior of primordial endothelial cells engaged in vasculogenesis, and they strongly suggest that endogenous VEGF is important in vascular patterning and regulation of vessel size (lumen formation). Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7543999

  1. Flavonoids from the leaves of Carya cathayensis Sarg. inhibit vascular endothelial growth factor-induced angiogenesis.

    PubMed

    Tian, Sha-Sha; Jiang, Fu-Sheng; Zhang, Kun; Zhu, Xue-Xin; Jin, Bo; Lu, Jin-Jian; Ding, Zhi-Shan

    2014-01-01

    The total flavonoids (TFs) were isolated from the leaves of Carya cathayensis Sarg. (LCC), a well-known Chinese medicinal herb commercially cultivated in Tianmu Mountain district, a cross area of Zhejiang and Anhui provinces in China. Five flavonoids, i.e. cardamonin, pinostrobin chalcone (PC), wogonin, chrysin, and pinocembrin were the main components of the TFs. The TFs and these pure compounds suppressed vascular endothelial growth factor (VEGF)-induced angiogenesis as detected in the mouse aortic ring assay, and cardamonin showed the best effect among them. To further elucidate the mechanisms for suppressing angiogenesis of these flavonoids, assays of VEGF-induced proliferation and migration in human umbilical vein endothelial cells (HUVECs) were performed. The TFs, cardamonin, pinocembrin, and chrysin obviously suppressed both VEGF-induced HUVEC proliferation and migration. However, PC and wogonin not only slightly inhibited VEGF-induced proliferation but also remarkably suppressed those of migration in HUVECs. Our further study showed that cardamonin decreased the phosphorylation of ERK and AKT induced by VEGF with a dose-dependent manner in HUVECs. Our findings indicate that the TFs and these pure flavonoids may become potential preventive and/or therapeutic agents against angiogenesis-related diseases. PMID:24096161

  2. Hepatic nerve growth factor induced by iron overload triggers defenestration in liver sinusoidal endothelial cells.

    PubMed

    Addo, Lynda; Tanaka, Hiroki; Yamamoto, Masayo; Toki, Yasumichi; Ito, Satoshi; Ikuta, Katsuya; Sasaki, Katsunori; Ohtake, Takaaki; Torimoto, Yoshihiro; Fujiya, Mikihiro; Kohgo, Yutaka

    2015-01-01

    The fenestrations of liver sinusoidal endothelial cells (LSECs) play important roles in the exchange of macromolecules, solutes, and fluid between blood and surrounding liver tissues in response to hepatotoxic drugs, toxins, and oxidative stress. As excess iron is a hepatotoxin, LSECs may be affected by excess iron. In this study, we found a novel link between LSEC defenestration and hepatic nerve growth factor (NGF) in iron-overloaded mice. By Western blotting, NGF was highly expressed, whereas VEGF and HGF were not, and hepatic NGF mRNA levels were increased according to digital PCR. Immunohistochemically, NGF staining was localized in hepatocytes, while TrkA, an NGF receptor, was localized in LSECs. Scanning electron microscopy revealed LSEC defenestration in mice overloaded with iron as well as mice treated with recombinant NGF. Treatment with conditioned medium from iron-overloaded primary hepatocytes reduced primary LSEC fenestrations, while treatment with an anti-NGF neutralizing antibody or TrkA inhibitor, K252a, reversed this effect. However, iron-loaded medium itself did not reduce fenestration. In conclusion, iron accumulation induces NGF expression in hepatocytes, which in turn leads to LSEC defenestration via TrkA. This novel link between iron and NGF may aid our understanding of the development of chronic liver disease. PMID:25460199

  3. Live cell imaging shows hepatocyte growth factor-induced Met dimerization.

    PubMed

    Koschut, David; Richert, Ludovic; Pace, Giuseppina; Niemann, Hartmut H; Mély, Yves; Orian-Rousseau, Véronique

    2016-07-01

    The canonical model of receptor tyrosine kinase (RTK) activation assumes that ligand-induced dimerization of inactive receptor monomers is a prerequisite for autophosphorylation. For several RTK families, recent results of fluorescence microscopy provided evidence for preformed receptor dimers that may or may not require ligand binding for kinase activity. Here we report, for the first time, the application of advanced quantitative fluorescence microscopy techniques to study changes in the oligomerization state of the RTK Met in response to stimulation by its endogenous ligand hepatocyte growth factor (HGF). We used inducible C-terminal fusions between Met and enhanced green fluorescent protein (EGFP) or red fluorescent protein (RFP) in combination with fluorescence resonance energy transfer (FRET)-based fluorescence-lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS). A small fraction of HGF-independent Met dimers appeared to be present in cells even at low receptor density. At high receptor density, both the fraction of Met dimers and the level of Met autophosphorylation increased in the absence of HGF. Stimulation with HGF at low receptor density significantly increased the fraction of Met dimers on live cells. We found no indications of Met oligomers larger than dimers. Our findings thus confirm a model of Met activation through HGF-induced dimerization and at the same time they support previous reports of Met dimers in unstimulated cells. The tools established in this work will be useful to further characterize the mechanism of Met activation and to define the contribution of co-receptors. PMID:27094128

  4. Occludin S490 Phosphorylation Regulates Vascular Endothelial Growth Factor-Induced Retinal Neovascularization.

    PubMed

    Liu, Xuwen; Dreffs, Alyssa; Díaz-Coránguez, Monica; Runkle, E Aaron; Gardner, Thomas W; Chiodo, Vince A; Hauswirth, William W; Antonetti, David A

    2016-09-01

    Occludin is a transmembrane tight junction protein that contributes to diverse cellular functions, including control of barrier properties, cell migration, and proliferation. Vascular endothelial growth factor (VEGF) induces phosphorylation of occludin at S490, which is required for VEGF-induced endothelial permeability. Herein, we demonstrate that occludin S490 phosphorylation also regulates VEGF-induced retinal endothelial cell proliferation and neovascularization. Using a specific antibody, phospho-occludin was located in centrosomes in endothelial cell cultures, animal models, and human surgical samples of retinal neovessels. Occludin S490 phosphorylation was found to increase with endothelial tube formation in vitro and in vivo during retinal neovascularization after induction of VEGF expression. More important, expression of occludin mutated at S490 to Ala, completely inhibited angiogenesis in cell culture models and in vivo. Collectively, these data suggest a novel role for occludin in regulation of endothelial proliferation and angiogenesis in a phosphorylation-dependent manner. These findings may lead to methods of regulating pathological neovascularization by specifically targeting endothelial cell proliferation. PMID:27423695

  5. Knockdown of stromal interaction molecule 1 attenuates hepatocyte growth factor-induced endothelial progenitor cell proliferation.

    PubMed

    Shi, Yankun; Song, Mingbao; Guo, Ruiwei; Wang, Hong; Gao, Pan; Shi, Weibin; Huang, Lan

    2010-03-01

    Increased Ca(2+) entry through store-operated Ca(2+) channels (SOCCs) plays an essential role in the regulation of hepatocyte growth factor (HGF)-induced cell proliferation. Stromal interaction molecule 1 (STIM1) is thought to transmit endoplasmic reticulum (ER) Ca(2+) store depletion signals to the plasma membrane (PM), causing the opening of SOCCs in the PM. However, the relationship between HGF and STIM1 in endothelial progenitor cell (EPC) proliferation remains uncharacterized. The objective of this study was to evaluate the potential involvement of STIM1 in HGF-induced EPC proliferation. For this purpose, we used cultured rat bone marrow-derived EPCs and found that HGF-induced EPC proliferation at low concentrations. Store-operated Ca(2+) entry (SOCE) was elevated in HGF-treated EPCs, and the SOCC inhibitors 2-aminoethoxydiphenyl borate (2-APB) and BTP-2 inhibited the HGF-induced proliferation response. Moreover, STIM1 mRNA and protein expression levels were increased in response to HGF stimulation and knockdown of STMI1 decreased SOCE and prevented HGF-induced EPC proliferation. In conclusion, our data suggest that HGF-induced EPC proliferation is mediated partly via activation of STIM1. PMID:20404049

  6. A growth factor-induced, spatially organizing cytoskeletal module enables rapid and persistent fibroblast migration

    PubMed Central

    Martin, Katrin; Vilela, Marco; Jeon, Noo Li; Danuser, Gaudenz; Pertz, Olivier

    2015-01-01

    Summary Directional migration requires robust front/back polarity. We find that fibroblasts treated with platelet-derived growth factor (PDGF) and pre-polarized by plating on a fibronectin line substrate, exhibit persistent migration for hours. This does not occur in the absence of PDGF, or on uniformly-coated fibronectin substrates. Persistent migration arises from establishment of two functional modules at cell front and back. At the front, formation of a zone containing podosome-like structures (PLS), dynamically correlates with low RhoA and myosin activity, and absence of a contractile lamella. At the back, myosin contractility specifically controls tail retraction with minimal crosstalk to the front module. The PLS zone is maintained in a dynamic steady state that preserves size and position relative to the cell front, allowing for long term coordination of front and back modules. We propose that front/back uncoupling achieved by the PLS zone is crucial for persistent migration in absence of directional cues. PMID:25268172

  7. Effects of angiopoietin-1 on vascular endothelial growth factor-induced angiogenesis in the mouse brain.

    PubMed

    Zhu, Y; Shwe, Y; Du, R; Chen, Y; Shen, F X; Young, W L; Yang, G Y

    2006-01-01

    A better understanding of angiogenic factors and their effects on angiogenesis in brain is necessary to treat cerebral vascular disorders such as ischemic brain injury. Vascular endothelial growth factor (VEGF) induces angiogenesis and increases blood-brain barrier (BBB) permeability in adult mouse brain. The effect of angiopoietin-1 on BBB leakage during the angiogenesis process is unclear. We sought to identify the effects of combining VEGF with angiopoietin-1 on cerebral angiogenesis and BBB. Adult male CD-1 mice underwent AdFc (adenoviral vector control), AdAng-1, VEGF protein, VEGF protein plus AdAng-1, or saline (negative control) injection. Brain microvessels were counted using lectin staining on tissue sections after 2 weeks of adenoviral gene transfer. The presence of zonula occludens-1 (ZO-1) was determined by Western blot analysis and immunohistochemistry. Microvessel count and augmented capillary diameter increased in mice treated with either VEGF protein or AdAng-1 plus VEGF protein compared to saline, AdFc, or AdAng-1 alone (p < 0.05). Double-labeled immunostaining demonstrated that ZO-1-positive staining was more complete on the microvessel wall in the AdAng-1 and AdAng-1 plus VEGF protein treated group compared to VEGF protein group. The results of ZO-1 expression from Western blot analysis paralleled that from immunohistochemistry (p < 0.05). We conclude that focal VEGF and angiopoietin-1 hyperstimulation in mouse brain increases microvessel density while maintaining ZO-1 protein expression, suggesting that angiopoietin-1 plays a role in synergistically inducing angiogenesis and BBB integrity. PMID:16671501

  8. Nerve growth factor induces sensitization of nociceptors without evidence for increased intraepidermal nerve fiber density.

    PubMed

    Hirth, Michael; Rukwied, Roman; Gromann, Alois; Turnquist, Brian; Weinkauf, Benjamin; Francke, Klaus; Albrecht, Philip; Rice, Frank; Hägglöf, Björn; Ringkamp, Matthias; Engelhardt, Maren; Schultz, Christian; Schmelz, Martin; Obreja, Otilia

    2013-11-01

    Nerve growth factor (NGF) is involved in the long-term sensitization of nociceptive processing linked to chronic pain. Functional and structural ("sprouting") changes can contribute. Thus, humans report long-lasting hyperalgesia to mechanical and electrical stimulation after intradermal NGF injection and NGF-induced sprouting has been reported to underlie cancer bone pain and visceral pain. Using a human-like animal model we investigated the relationship between the structure and function of unmyelinated porcine nociceptors 3 weeks after intradermal NGF treatment. Axonal and sensory characteristics were studied by in vivo single-fiber electrophysiology and immunohistochemistry. C fibers recorded extracellularly were classified based on mechanical response and activity-dependent slowing (ADS) of conduction velocity. Intraepidermal nerve fiber (IENF) densities were assessed by immunohistochemistry in pigs and in human volunteers using the same NGF model. NGF increased conduction velocity and reduced ADS and propagation failure in mechano-insensitive nociceptors. The proportion of mechano-sensitive C nociceptors within NGF-treated skin areas increased from 45.1% (control) to 71% and their median mechanical thresholds decreased from 40 to 20 mN. After NGF application, the mechanical receptive fields of nociceptors increased from 25 to 43 mm(2). At the structural level, however, IENF density was not increased by NGF. In conclusion, intradermal NGF induces long-lasting axonal and mechanical sensitization in porcine C nociceptors that corresponds to hyperalgesia observed in humans. Sensitization is not accompanied by increased IENF density, suggesting that NGF-induced hyperalgesia might not depend on changes in nerve fiber density but could be linked to the recruitment of previously silent nociceptors. PMID:23891896

  9. Nerve growth factor alters microtubule targeting agent-induced neurotransmitter release but not MTA-induced neurite retraction in sensory neurons.

    PubMed

    Pittman, Sherry K; Gracias, Neilia G; Fehrenbacher, Jill C

    2016-05-01

    Peripheral neuropathy is a dose-limiting side effect of anticancer treatment with the microtubule-targeted agents (MTAs), paclitaxel and epothilone B (EpoB); however, the mechanisms by which the MTAs alter neuronal function and morphology are unknown. We previously demonstrated that paclitaxel alters neuronal sensitivity, in vitro, in the presence of nerve growth factor (NGF). Evidence in the literature suggests that NGF may modulate the neurotoxic effects of paclitaxel. Here, we examine whether NGF modulates changes in neuronal sensitivity and morphology induced by paclitaxel and EpoB. Neuronal sensitivity was assessed using the stimulated release of calcitonin gene-related peptide (CGRP), whereas morphology of established neurites was evaluated using a high content screening system. Dorsal root ganglion cultures, maintained in the absence or presence of NGF, were treated from day 7 to day 12 in culture with paclitaxel (300nM) or EpoB (30nM). Following treatment, the release of CGRP was stimulated using capsaicin or high extracellular potassium. In the presence of NGF, EpoB mimicked the effects of paclitaxel: capsaicin-stimulated release was attenuated, potassium-stimulated release was slightly enhanced and the total peptide content was unchanged. In the absence of NGF, both paclitaxel and EpoB decreased capsaicin- and potassium-stimulated release and the total peptide content, suggesting that NGF may reverse MTA-induced hyposensitivity. Paclitaxel and EpoB both decreased neurite length and branching, and this attenuation was unaffected by NGF in the growth media. These differential effects of NGF on neuronal sensitivity and morphology suggest that neurite retraction is not a causative factor to alter neuronal sensitivity. PMID:26883566

  10. Neurite growth acceleration of adult Dorsal Root Ganglion neurons illuminated by low-level Light Emitting Diode light at 645 nm.

    PubMed

    Burland, Marion; Paris, Lambert; Quintana, Patrice; Bec, Jean-Michel; Diouloufet, Lucie; Sar, Chamroeun; Boukhaddaoui, Hassan; Charlot, Benoit; Braga Silva, Jefferson; Chammas, Michel; Sieso, Victor; Valmier, Jean; Bardin, Fabrice

    2015-06-01

    The effect of a 645 nm Light Emitting Diode (LED) light irradiation on the neurite growth velocity of adult Dorsal Root Ganglion (DRG) neurons with peripheral axon injury 4-10 days before plating and without previous injury was investigated. The real amount of light reaching the neurons was calculated by taking into account the optical characteristics of the light source and of media in the light path. The knowledge of these parameters is essential to be able to compare results of the literature and a way to reduce inconsistencies. We found that 4 min irradiation of a mean irradiance of 11.3 mW/cm(2) (corresponding to an actual irradiance reaching the neurons of 83 mW/cm(2)) induced a 1.6-fold neurite growth acceleration on non-injured neurons and on axotomized neurons. Although the axotomized neurons were naturally already in a rapid regeneration process, an enhancement was found to occur while irradiating with the LED light, which may be promising for therapy applications. Dorsal Root Ganglion neurons (A) without previous injury and (B) subjected to a conditioning injury. PMID:25077453

  11. The effects of gradients of nerve growth factor immobilized PCLA scaffolds on neurite outgrowth in vitro and peripheral nerve regeneration in rats.

    PubMed

    Tang, Shuo; Zhu, Jixiang; Xu, Yangbin; Xiang, Andy Peng; Jiang, Mei Hua; Quan, Daping

    2013-09-01

    Introducing concentration gradients of nerve growth factor (NGF) into conduits for repairing of peripheral nerve injury is crucial for nerve regeneration and guidance. Herein, combining differential adsorption of NGF/silk fibroin (SF) coating, the gradient of NGF-immobilized membranes (G-Ms) and nanofibrous nerve conduits (G-nNCs) were successfully fabricated. The efficacy of NGF gradients was confirmed by a quantitative comparison of dorsal root ganglia (DRG) neurite outgrowth on the G-Ms or uniform NGF-immobilized membranes (U-Ms). Significantly, the neurite turning ratio was 0.48 ± 0.11 for G-M group, but it was close to zero for U-M group. The neurite length of DRGs in the middle of the G-Ms was significantly longer than that of U-M group, even though the average NGF concentration was approximated. Furthermore, 12 weeks after implantation in rats with a 14 mm gap of sciatic nerve injury, G-nNCs achieved satisfying outcomes of nerve regeneration associated with morphological and functional improvements, which was superior to that of the uniform NGF-immobilized nNCs (U-nNCs). Sciatic function index (SFI), compound muscle action potentials (CMAPs), total number of myelinated nerve fibers, thickness of myelin sheath were similar for the G-nNCs and autografts, with the G-nNCs having a higher density of axons than the autografts. Our results demonstrated the significant role of introducing NGF gradients into scaffolds in promoting nerve regeneration. PMID:23791502

  12. Olfactory ensheathing cell-neurite alignment enhances neurite outgrowth in scar-like cultures

    PubMed Central

    Khankan, Rana R.; Wanner, Ina B.; Phelps, Patricia E.

    2015-01-01

    The regenerative capacity of the adult CNS neurons after injury is strongly inhibited by the spinal cord lesion site environment that is composed primarily of the reactive astroglial scar and invading meningeal fibroblasts. Olfactory ensheathing cell (OEC) transplantation facilitates neuronal survival and functional recovery after a complete spinal cord transection, yet the mechanisms by which this recovery occurs remain unclear. We used a unique multicellular scar-like culture model to test if OECs promote neurite outgrowth in growth inhibitory areas. Astrocytes were mechanically injured and challenged by meningeal fibroblasts to produce key inhibitory elements of a spinal cord lesion. Neurite outgrowth of postnatal cerebral cortical neurons was assessed on three substrates: quiescent astrocyte control cultures, reactive astrocyte scar-like cultures, and scar-like cultures with OECs. Initial results showed that OECs enhanced total neurite outgrowth of cortical neurons in a scar-like environment by 60%. We then asked if the neurite growth-promoting properties of OECs depended on direct alignment between neuronal and OEC processes. Neurites that aligned with OECs were nearly three times longer when they grew on inhibitory meningeal fibroblast areas and twice as long on reactive astrocyte zones compared to neurites not associated with OECs. Our results show that OECs can independently enhance neurite elongation and that direct OEC-neurite cell contact can provide a permissive substrate that overcomes the inhibitory nature of the reactive astrocyte scar border and the fibroblast-rich spinal cord lesion core. PMID:25863021

  13. Dendrite and Axon Specific Geometrical Transformation in Neurite Development

    PubMed Central

    Mironov, Vasily I.; Semyanov, Alexey V.; Kazantsev, Victor B.

    2016-01-01

    We propose a model of neurite growth to explain the differences in dendrite and axon specific neurite development. The model implements basic molecular kinetics, e.g., building protein synthesis and transport to the growth cone, and includes explicit dependence of the building kinetics on the geometry of the neurite. The basic assumption was that the radius of the neurite decreases with length. We found that the neurite dynamics crucially depended on the relationship between the rate of active transport and the rate of morphological changes. If these rates were in the balance, then the neurite displayed axon specific development with a constant elongation speed. For dendrite specific growth, the maximal length was rapidly saturated by degradation of building protein structures or limited by proximal part expansion reaching the characteristic cell size. PMID:26858635

  14. The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis

    PubMed Central

    Nelson, Michaela; Millican-Slater, Rebecca; Forrest, Lorna C; Brackenbury, William J

    2014-01-01

    Voltage-gated Na+ channels (VGSCs) are heteromeric proteins composed of pore-forming α subunits and smaller β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). β1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. β1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/β1 protein were up-regulated in BCa specimens, compared with normal breast tissue. β1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. β1 over-expression also increased vascularization and reduced apoptosis in the primary tumours, and β1 over-expressing tumour cells had an elongate morphology. In vitro, β1 potentiated outgrowth of processes from BCa cells co-cultured with fibroblasts, via trans-homophilic adhesion. β1-mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na+ current, thus replicating the mechanism by which β1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, β1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for β1 in tumour growth and metastasis in vivo. We propose that β1 warrants further study as a potential biomarker and targeting β1-mediated adhesion interactions may have value as a novel anti-cancer therapy. PMID:24729314

  15. Patient Mutations of the Intellectual Disability Gene KDM5C Downregulate Netrin G2 and Suppress Neurite Growth in Neuro2a Cells.

    PubMed

    Wei, Gengze; Deng, Xinxian; Agarwal, Saurabh; Iwase, Shigeki; Disteche, Christine; Xu, Jun

    2016-09-01

    The X-linked lysine (K)-specific demethylase 5C (KDM5C) gene plays an important role in brain development and behavior. It encodes a histone demethylase that is involved in gene regulation in neuronal differentiation and morphogenesis. When mutated, it causes neuropsychiatric symptoms, such as intellectual disability, delayed language development, epilepsy, and impulsivity. To better understand how the patient mutations affect neuronal development, we expressed KDM5C mutants in Neuro2a cells, a mouse neuroblastoma cell line. Retinoic acid (RA)-induced neurite growth was suppressed by the mutation KDM5C (Y751C) , KDM5C (H514A) , and KDM5C (F642L) , but not KDM5C (D87G) or KDM5C (A388P) . RNA-seq analysis indicated an upregulation of genes important for neuronal development, such as Ntng2, Enah, Gas1, Slit2, and Dscam, in response to the RA treatment in control Neuro2a cells transfected with GFP or wild-type KDM5C. In contrast, in cells transfected with KDM5C (Y751C) , these genes were not upregulated by RA. Ntng2 was downregulated in cells with KDM5C mutations, concordant with the lower levels of H3K4 methylation at its promoter. Moreover, knocking down Ntng2 in control Neuro2a cells led to the phenotype of short neurites similar to that of cells with KDM5C (Y751C) , whereas Ntng2 overexpression in the mutant cells rescued the morphological phenotype. These findings provide new insight into the pathogenesis of phenotypes associated with KDM5C mutations. PMID:27421841

  16. Real-time detection of neurite outgrowth using microfluidic device

    NASA Astrophysics Data System (ADS)

    Kim, Samhwan; Jang, Jongmoon; Choi, Hongsoo; Moon, Cheil

    2013-05-01

    We developed a simple method for real-time detection of the neurite outgrowth using microfluidic device. Our microfluidic device contains three compartmentalized channels which are for cell seeding, hydrogel and growth factors. Collagen gel is filled in the middle channel and pheochromocytoma (PC12) cells are seeded in the left channel. To induce differentiation of PC12 cells, 50 ng/ml to1000 ng/ml of nerve growth factor (NGF) is introduced into the right channel. After three days of NGF treatment, PC12 cells begin to extend neurites and formed neurite network from sixth day. Quantification of neurite outgrowth is analyzed by measuring the total area of neurites. On sixth day, the area is doubled compared to the area on third day and increases by 20 times on ninth day.

  17. Myelin from MAG-deficient mice is a strong inhibitor of neurite outgrowth.

    PubMed

    Ng, W P; Cartel, N; Li, C; Roder, J; Lozano, A

    1996-03-22

    Myelin-associated glycoprotein (MAG) has potent neurite outgrowth inhibitory activity in vitro. To assess the importance of MAG in the neurite outgrowth inhibitory activity in CNS myelin, we used an in vitro bioassay to characterize neurite growth on CNS myelin derived from mice carrying a null mutation of the MAG gene. Myelin proteins from MAG-deficient mice inhibited neurite outgrowth to a similar degree to the wild-type CNS myelin. These results suggest that CNS myelin molecules other than MAG exert strong inhibitory effects on the growth of neurites. PMID:8724661

  18. Morphological identification and development of neurite in Drosophila ventral nerve cord neuropil.

    PubMed

    Gan, Guangming; Lv, Huihui; Xie, Wei

    2014-01-01

    In Drosophila, ventral nerve cord (VNC) occupies most of the larval central nervous system (CNS). However, there is little literature elaborating upon the specific types and growth of neurites as defined by their structural appearance in Drosophila larval VNC neuropil. Here we report the ultrastructural development of different types VNC neurites in ten selected time points in embryonic and larval stages utilizing transmission electron microscopy. There are four types of axonal neurites as classified by the type of vesicular content: clear vesicle (CV) neurites have clear vesicles and some T-bar structures; Dense-core vesicle (DV) neurites have dense-core vesicles and without T-bar structures; Mixed vesicle (MV) neurites have mixed vesicles and some T-bar structures; Large vesicle (LV) neurites are dominated by large, translucent spherical vesicles but rarely display T-bar structures. We found dramatic remodeling in CV neurites which can be divided into five developmental phases. The neurite is vacuolated in primary (P) phase, they have mitochondria, microtubules or big dark vesicles in the second (S) phase, and they contain immature synaptic features in the third (T) phase. The subsequent bifurcate (B) phase appears to undergo major remodeling with the appearance of the bifurcation or dendritic growth. In the final mature (M) phase, high density of commensurate synaptic vesicles are distributed around T-bar structures. There are four kinds of morphological elaboration of the CVI neurite sub-types. First, new neurite produces at the end of axon. Second, new neurite bubbles along the axon. Third, the preexisting neurite buds and develops into several neurites. The last, the bundled axons form irregularly shape neurites. Most CVI neurites in M phase have about 1.5-3 µm diameter, they could be suitable to analyze their morphology and subcellular localization of specific proteins by light microscopy, and they could serve as a potential model in CNS in vivo development

  19. Tax and Semaphorin 4D Released from Lymphocytes Infected with Human Lymphotropic Virus Type 1 and Their Effect on Neurite Growth.

    PubMed

    Quintremil, Sebastián; Alberti, Carolina; Rivera, Matías; Medina, Fernando; Puente, Javier; Cartier, Luis; Ramírez, Eugenio; Tanaka, Yuetsu; Valenzuela, M Antonieta

    2016-01-01

    Human lymphotropic virus type 1 (HTLV-1) is a retrovirus causing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a neurodegenerative central nervous system (CNS) axonopathy. This virus mainly infects CD4(+) T lymphocytes without evidence of neuronal infection. Viral Tax, secreted from infected lymphocytes infiltrated in the CNS, is proposed to alter intracellular pathways related to axonal cytoskeleton dynamics, producing neurological damage. Previous reports showed a higher proteolytic release of soluble Semaphorin 4D (sSEMA-4D) from CD4(+) T cells infected with HTLV-1. Soluble SEMA-4D binds to its receptor Plexin-B1, activating axonal growth collapse pathways in the CNS. In the current study, an increase was found in both SEMA-4D in CD4(+) T cells and sSEMA-4D released to the culture medium of peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients compared to asymptomatic carriers and healthy donors. After a 16-h culture, infected PBMCs showed significantly higher levels of CRMP-2 phosphorylated at Ser(522). The effect was blocked either with anti-Tax or anti-SEMA-4D antibodies. The interaction of Tax and sSEMA-4D was found in secreted medium of PBMCs in patients, which might be associated with a leading role of Tax with the SEMA-4D-Plexin-B1 signaling pathway. In infected PBMCs, the migratory response after transwell assay showed that sSEMA-4D responding cells were CD4(+)Tax(+) T cells with a high CRMP-2 pSer(522) content. In the present study, the participation of Tax-sSEMA-4D in the reduction in neurite growth in PC12 cells produced by MT2 (HTLV-1-infected cell line) culture medium was observed. These results lead to the participation of plexins in the reported effects of infected lymphocytes on neuronal cells. PMID:26389656

  20. Regulation by intracellular Ca sup 2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis

    SciTech Connect

    Miyata, Yoshihiko Tokyo Metropolitan Inst. of Medical Science ); Nishida, Eisuke; Sakai, Hikoichi ); Koyasu, Shigeo; Yahara, Ichiro )

    1989-04-01

    Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes and stimulate the fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells. An increase in intracellular Ca{sup 2+} concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca{sup 2+} or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca{sup 2+} and cAMP. {sup 125}I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca{sup 2+}- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.

  1. Reactive Oxygen Species, Ki-Ras, and Mitochondrial Superoxide Dismutase Cooperate in Nerve Growth Factor-induced Differentiation of PC12 Cells*

    PubMed Central

    Cassano, Silvana; Agnese, Savina; D'Amato, Valentina; Papale, Massimo; Garbi, Corrado; Castagnola, Patrizio; Ruocco, Maria Rosaria; Castellano, Immacolata; De Vendittis, Emmanuele; Santillo, Mariarosaria; Amente, Stefano; Porcellini, Antonio; Avvedimento, Enrico Vittorio

    2010-01-01

    Nerve growth factor (NGF) induces terminal differentiation in PC12, a pheochromocytoma-derived cell line. NGF binds a specific receptor on the membrane and triggers the ERK1/2 cascade, which stimulates the transcription of neural genes. We report that NGF significantly affects mitochondrial metabolism by reducing mitochondrial-produced reactive oxygen species and stabilizing the electrochemical gradient. This is accomplished by stimulation of mitochondrial manganese superoxide dismutase (MnSOD) both transcriptionally and post-transcriptionally via Ki-Ras and ERK1/2. Activation of MnSOD is essential for completion of neuronal differentiation because 1) expression of MnSOD induces the transcription of a neuronal specific promoter and neurite outgrowth, 2) silencing of endogenous MnSOD by small interfering RNA significantly reduces transcription induced by NGF, and 3) a Ki-Ras mutant in the polylysine stretch at the COOH terminus, unable to stimulate MnSOD, fails to induce complete differentiation. Overexpression of MnSOD restores differentiation in cells expressing this mutant. ERK1/2 is also downstream of MnSOD, as a SOD mimetic drug stimulates ERK1/2 with the same kinetics of NGF and silencing of MnSOD reduces NGF-induced late ERK1/2. Long term activation of ERK1/2 by NGF requires SOD activation, low levels of hydrogen peroxide, and the integrity of the microtubular cytoskeleton. Confocal immunofluorescence shows that NGF stimulates the formation of a complex containing membrane-bound Ki-Ras, microtubules, and mitochondria. We propose that active NGF receptor induces association of mitochondria with plasma membrane. Local activation of ERK1/2 by Ki-Ras stimulates mitochondrial SOD, which reduces reactive oxygen species and produces H2O2. Low and spatially restricted levels of H2O2 induce and maintain long term ERK1/2 activity and ultimately differentiation of PC12 cells. PMID:20495008

  2. Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors.

    PubMed

    Lobo, M V T; Arenas, M I; Huerta, L; Sacristán, S; Pérez-Crespo, M; Gutiérrez-Adán, A; Díaz-Gil, J J; Lasunción, M A; Martín-Hidalgo, A

    2015-01-15

    The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3β-hydroxysteroid dehydrogenase (3β-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3β-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery. PMID:25389365

  3. Epidermal Growth Factor-Induced Tumor Cell Invasion and Metastasis Initiated by Dephosphorylation and Downregulation of Focal Adhesion Kinase

    PubMed Central

    Lu, Zhimin; Jiang, Guoqiang; Blume-Jensen, Peter; Hunter, Tony

    2001-01-01

    Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis. PMID:11359909

  4. Epidermal growth factor-induced stimulation of proliferation and gene expression changes in the hypotrichous ciliate, Stylonychia lemnae.

    PubMed

    Mu, Weijie; Wang, Qi; Bourland, William A; Jiang, Chuanqi; Yuan, Dongxia; Pan, Xuming; Miao, Wei; Chen, Ying; Xiong, Jie

    2016-10-30

    Epidermal growth factor (EGF) induces proliferation of epidermal and epithelial tissues in mammals. However, the effect of EGF on the single-celled eukaryotes is not well characterized, especially in the protists. Ciliates, an important group of protists, are well characterized as both pollution indicators and model organisms for research. Stylonychia lemnae, is one of the most common free-living ciliates, widely distributed in ponds, rivers and marshes. Here, we report the role of EGF on cell proliferation stimulation in S. lemnae. The growth curve of S. lemnae was established, and the stimulation effect of EGF on the proliferation of S. lemnae was investigated. Based on the results, potential EGF receptors were identified in S. lemnae according to the conserved domains and gene expression. Differential gene expression revealed that EGF-induced genes in other organisms (e.g. antioxidant) also up-regulated in S. lemnae cells at propagation stages. In addition, our results showed that EGF could up-regulate the signal transduction-related processes in the decline stage of S. lemnae cells, indicating its potential function in apoptosis inhibition. In summary, this study reports findings of the first investigation of EGF effects in hypotrich ciliates, and establishes an additional system for the study of the molecular mechanisms of EGF actions in eukaryotic cell division and proliferation. PMID:27506312

  5. Platelet-released growth factors induce the antimicrobial peptide human beta-defensin-2 in primary keratinocytes.

    PubMed

    Bayer, Andreas; Lammel, Justus; Rademacher, Franziska; Groß, Justus; Siggelkow, Markus; Lippross, Sebastian; Klüter, Tim; Varoga, Deike; Tohidnezhad, Mersedeh; Pufe, Thomas; Cremer, Jochen; Gläser, Regine; Harder, Jürgen

    2016-06-01

    Platelet-released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet-rich fibrin (PRF(®) )] are thrombocyte concentrate lysates that support healing of chronic, hard-to-heal and infected wounds. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound-related bacteria. In this study, we analysed the influence of PRGF on hBD-2 expression in human primary keratinocytes and the influence of Vivostat PRF(®) on hBD-2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD-2 gene and protein expressions in a concentration- and time-dependent manner. The use of blocking antibodies revealed that the PRGF-mediated hBD-2 induction was partially mediated by the epidermal growth factor receptor and the interleukin-6 receptor (IL-6R). Luciferase gene reporter assays indicated that the hBD-2 induction through PRGF required activation of the transcription factor activator protein 1 (AP-1), but not of NF-kappaB. In concordance with these cell culture data, Vivostat PRF(®) induced hBD-2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD-2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds. PMID:26843467

  6. Keratinocyte Growth Factor Induces Gene Expression Signature Associated with Suppression of Malignant Phenotype of Cutaneous Squamous Carcinoma Cells

    PubMed Central

    Toriseva, Mervi; Ala-aho, Risto; Peltonen, Sirkku; Peltonen, Juha; Grénman, Reidar; Kähäri, Veli-Matti

    2012-01-01

    Keratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells. The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair. However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known. We have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin. The expression of KGF receptor (KGFR) mRNA was lower in cutaneous SCCs (n = 6) than in normal skin samples (n = 6). Expression of KGFR mRNA was detected in 6 out of 8 cutaneous SCC cell lines and the levels were downregulated by 24-h treatment with KGF. KGF did not stimulate SCC cell proliferation, but it reduced invasion of SCC cells through collagen. Gene expression profiling of three cutaneous SCC cell lines treated with KGF for 24 h revealed a specific gene expression signature characterized by upregulation of a set of genes specifically downregulated in SCC cells compared to normal epidermal keratinocytes, including genes with tumor suppressing properties (SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1). KGF also induced downregulation of a set of genes specifically upregulated in SCC cells compared to normal keratinocytes, including genes associated with tumor progression (MMP13, MATN2, CXCL10, and IGFBP3). Downregulation of MMP-13 and KGFR expression in SCC cells and HaCaT cells was mediated via ERK1/2. Activation of ERK1/2 in HaCaT cells and tumorigenic Ha-ras-transformed HaCaT cells resulted in downregulation of MMP-13 and KGFR expression. These results provide evidence, that KGF does not promote progression of cutaneous SCC, but rather suppresses the malignant phenotype of cutaneous SCC cells by regulating the expression of several genes differentially expressed in SCC cells, as compared to normal keratinocytes. PMID:22427941

  7. Vascular endothelial growth factor-induced elimination of the type 1 interferon receptor is required for efficient angiogenesis.

    PubMed

    Zheng, Hui; Qian, Juan; Carbone, Christopher J; Leu, N Adrian; Baker, Darren P; Fuchs, Serge Y

    2011-10-01

    Angiogenesis is stimulated by vascular endothelial growth factor (VEGF) and antagonized by type 1 interferons, including IFN-α/β. On engaging their respective receptors (VEGFR2 and IFNAR), both stimuli activate protein kinase D2 (PKD2) and type 1 IFNs require PKD2 activation and recruitment to IFNAR1 to promote the phosphorylation-dependent ubiquitination, down-regulation, and degradation of the cognate receptor chain, IFNAR1. Data reveal that PKD2 activity is dispensable for VEGF-stimulated down-regulation of VEGFR2. Remarkably, VEGF treatment promotes the recruitment of PKD2 to IFNAR1 as well as ensuing phosphorylation, ubiquitination, and degradation of IFNAR1. In cells exposed to VEGF, phosphorylation-dependent degradation of IFNAR1 leads to an inhibition of type 1 IFN signaling and is required for efficient VEGF-stimulated angiogenesis. Importance of this mechanism for proangiogenic or antiangiogenic responses in cells exposed to counteracting stimuli and the potential medical significance of this regulation are discussed. PMID:21832278

  8. Nerve growth factor induces facial heat hyperalgesia and plays a role in trigeminal neuropathic pain in rats.

    PubMed

    Dos Reis, Renata C; Kopruszinski, Caroline M; Nones, Carina F M; Chichorro, Juliana G

    2016-09-01

    There is preclinical evidence that nerve growth factor (NGF) contributes toward inflammatory hyperalgesia in the orofacial region, but the mechanisms underlying its hyperalgesic effect as well as its role in trigeminal neuropathic pain require further investigation. This study investigated the ability of NGF to induce facial heat hyperalgesia and the involvement of tyrosine kinase receptor A, transient receptor potential vanilloid 1, and mast cells in NGF pronociceptive effects. In addition, the role of NGF in heat hyperalgesia in a model of trigeminal neuropathic pain was evaluated. NGF injection into the upper lip of naive rats induced long-lasting heat hyperalgesia. Pretreatment with an antibody anti-NGF, antagonists of tyrosine kinase receptor A, and transient receptor potential vanilloid 1 receptors or compound 48/80, to induce mast-cell degranulation, all attenuated NGF-induced hyperalgesia. In a rat model of trigeminal neuropathic pain, local treatment with anti-NGF significantly reduced heat hyperalgesia. In addition, increased NGF levels were detected in the ipsilateral infraorbital nerve branch at the time point that represents the peak of heat hyperalgesia. The results suggest that NGF is a prominent hyperalgesic mediator in the trigeminal system and it may represent a potential therapeutic target for the management of painful orofacial conditions, including trigeminal neuropathic pain. PMID:27392124

  9. Essential Role of Transglutaminase 2 in Vascular Endothelial Growth Factor-Induced Vascular Leakage in the Retina of Diabetic Mice.

    PubMed

    Lee, Yeon-Ju; Jung, Se-Hui; Kim, Su-Hyeon; Kim, Min-Soo; Lee, Sungeun; Hwang, JongYun; Kim, Soo-Youl; Kim, Young-Myeong; Ha, Kwon-Soo

    2016-08-01

    Diabetic retinopathy is predominantly caused by vascular endothelial growth factor (VEGF)-induced vascular leakage; however, the underlying mechanism is unclear. Here we designed an in vivo transglutaminase (TGase) activity assay in mouse retina and demonstrated that hyperglycemia induced vascular leakage by activating TGase2 in diabetic retina. VEGF elevated TGase2 activity through sequential elevation of intracellular Ca(2+) and reactive oxygen species (ROS) concentrations in endothelial cells. The TGase inhibitors cystamine and monodansylcadaverin or TGase2 small interfering RNA (siRNA) prevented VEGF-induced stress fiber formation and vascular endothelial (VE)-cadherin disruption, which play a critical role in modulating endothelial permeability. Intravitreal injection of two TGase inhibitors or TGase2 siRNA successfully inhibited hyperglycemia-induced TGase activation and microvascular leakage in the retinas of diabetic mice. C-peptide or ROS scavengers also inhibited TGase activation in diabetic mouse retinas. The role of TGase2 in VEGF-induced vascular leakage was further supported using diabetic TGase2(-/-) mice. Thus, our findings suggest that ROS-mediated activation of TGase2 plays a key role in VEGF-induced vascular leakage by stimulating stress fiber formation and VE-cadherin disruption. PMID:27207524

  10. MET inhibitor PHA-665752 suppresses the hepatocyte growth factor-induced cell proliferation and radioresistance in nasopharyngeal carcinoma cells

    SciTech Connect

    Liu, Tongxin; Li, Qi; Sun, Quanquan; Zhang, Yuqin; Yang, Hua; Wang, Rong; Chen, Longhua; Wang, Wei

    2014-06-20

    Highlights: • We demonstrated that irradiation induced MET overexpression and activation. • The aberrant MET signal mediated by HGF induced proliferation and radioresistance of NPC cells. • MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. • PHA-665752 suppressed the three downstream pathway of HGF/MET signal in a dose-dependent manner. - Abstract: Although ionizing radiation (IR) has provided considerable improvements in nasopharyngeal carcinoma (NPC), in subsets of patients, radioresistance is still a major problem in the treatment. In this study, we demonstrated that irradiation induced MET overexpression and activation, and the aberrant MET signal mediated by hepatocyte growth factor (HGF) induced radioresistance. We also found that MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. Further investigation indicated that PHA-665752 suppressed the phosphorylation of the Akt, ERK1/2, and STAT3 proteins in a dose-dependent manner. Our data indicated that the combination of IR with a MET inhibitor, such as PHA-665752, might be a promising therapeutic strategy for NPC.

  11. Proangiogenic role of ephrinB1/EphB1 in basic fibroblast growth factor-induced corneal angiogenesis.

    PubMed

    Kojima, Takashi; Chang, Jin-Hong; Azar, Dimitri T

    2007-02-01

    Corneal neovascularization is a vision-threatening condition caused by various ocular pathological conditions. The aim of this study was to evaluate the function of the ephrin ligands and Eph receptors in vitro and in vivo in corneal angiogenesis in a mouse model. The Eph tyrosine kinase receptors and their ligands, ephrins, are expressed on the cell surface. The functions of Eph and ephrins have been shown to regulate axonal guidance, segmentation, cell migration, and angiogenesis. Understanding the roles of Eph and ephrin in corneal angiogenesis may provide a therapeutic intervention for the treatment of angiogenesis-related disorders. Immunohistochemical studies demonstrated that ephrinB1 and EphB1 were expressed in basic fibroblast growth factor (bFGF)-induced vascularized corneas. EphB1 was specifically colocalized with vascular endothelial marker CD31 surrounded by type IV collagen. EphrinB1 was expressed in corneal-resident keratocytes and neutrophils. Recombinant ephrinB1-Fc, which induces EphB receptor activation, enhanced bFGF-induced tube formation in an in vitro aortic ring assay and promoted bFGF-induced corneal angiogenesis in vivo in a corneal pocket assay. Synergistically enhanced and sustained activation of extracellular signal-regulated kinase was noted in vascular endothelial cell lines after stimulation with ephrin B1 and bFGF combinations. These results suggest that ephrinB1 plays a synergistic role in corneal neovascularization. PMID:17255342

  12. Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation

    PubMed Central

    Hsu, Jinn-Yuan; Chang, Kwang-Yu; Chen, Shang-Hung; Lee, Chung-Ta; Chang, Sheng-Tsung; Cheng, Hung-Chi; Chang, Wen-Chang; Chen, Ben-Kuen

    2015-01-01

    Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of cyclooxygenase-2 (COX-2) mediates EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced COX-2 expression mainly in HNSCC. The tumor cell transformation induced by EGF was repressed by COX-2 knockdown, and this repression was reversed by simultaneously treating the cells with EGF and prostaglandin E2 (PGE2). The down-regulation of COX-2 expression or inhibition of COX-2 activity significantly blocked EGF enhancement of cell migration and invasion, but the addition of PGE2 compensated for this inhibitory effect in COX-2-knockdown cells. COX-2 depletion inhibited EGF-induced matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. The inhibitory effect of COX-2 depletion on MMPs and the fibronectin/Rac1/cdc42 axis were reversed by co-treatment with PGE2. Furthermore, depletion of fibronectin impeded the COX-2-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastatic seeding of the lungs. These results demonstrate that EGF-induced COX-2 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of COX-2 expression and activation may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis. PMID:25595899

  13. Matrix interactions modulate neurotrophin-mediated neurite outgrowth and pathfinding

    PubMed Central

    Madl, Christopher M.; Heilshorn, Sarah C.

    2015-01-01

    Both matrix biochemistry and neurotrophic factors are known to modulate neurite outgrowth and pathfinding; however, the interplay between these two factors is less studied. While previous work has shown that the biochemical identity of the matrix can alter the outgrowth of neurites in response to neurotrophins, the importance of the concentration of cell-adhesive ligands is unknown. Using engineered elastin-like protein matrices, we recently demonstrated a synergistic effect between matrix-bound cell-adhesive ligand density and soluble nerve growth factor treatment on neurite outgrowth from dorsal root ganglia. This synergism was mediated by Schwann cell-neurite contact through L1CAM. Cell-adhesive ligand density was also shown to alter the pathfinding behavior of dorsal root ganglion neurites in response to a gradient of nerve growth factor. While more cell-adhesive matrices promoted neurite outgrowth, less cell-adhesive matrices promoted more faithful neurite pathfinding. These studies emphasize the importance of considering both matrix biochemistry and neurotrophic factors when designing biomaterials for peripheral nerve regeneration. PMID:26170800

  14. Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGF-Stat5 DNA binding activity.

    PubMed Central

    Gouilleux, F; Pallard, C; Dusanter-Fourt, I; Wakao, H; Haldosen, L A; Norstedt, G; Levy, D; Groner, B

    1995-01-01

    The molecular components which mediate cytokine signaling from the cell membrane to the nucleus were studied. Upon the interaction of cytokines with their receptors, members of the janus kinase (Jak) family of cytoplasmic protein tyrosine kinases and of the signal transducers and activators of transcription (Stat) family of transcription factors are activated through tyrosine phosphorylation. It has been suggested that the Stat proteins are substrates of the Jak protein tyrosine kinases. MGF-Stat5 is a member of the Stat family which has been found to confer the prolactin response. MGF-Stat5 can be phosphorylated and activated in its DNA binding activity by Jak2. The activation of MGF-Stat5 is not restricted to prolactin. Erythropoietin (EPO) and growth hormone (GH) stimulate the DNA binding activity of MGF-Stat5 in COS cells transfected with vectors encoding EPO receptor and MGF-Stat5 or vectors encoding GH receptor and MGF-Stat5. The activation of DNA binding by prolactin, EPO and GH requires the phosphorylation of tyrosine residue 694 of MGF-Stat5. The transcriptional induction of a beta-casein promoter luciferase construct in transiently transfected COS cells is specific for the prolactin activation of MGF-Stat5; it is not observed in EPO- and GH-treated cells. In the UT7 human hematopoietic cell line, EPO and granulocyte-macrophage colony stimulating factor activate the DNA binding activity of a factor closely related to MGF-Stat5 with respect to its immunological reactivity, DNA binding specificity and molecular weight. These results suggest that MGF-Stat5 regulates physiological processes in mammary epithelial cells, as well as in hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:7744007

  15. Involvement of PI3K and ERK1/2 pathways in hepatocyte growth factor-induced cholangiocarcinoma cell invasion

    PubMed Central

    Menakongka, Apaporn; Suthiphongchai, Tuangporn

    2010-01-01

    AIM: To investigate the role of hepatocyte growth factor (HGF) in cholangiocarcinoma (CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS: Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines, HuCCA-1 and KKU-M213, using Transwell in vitro assay. Levels of proteins of interest and their phosphorylated forms were determined by Western blotting. Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS: Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line. HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization, but did not affect the levels of secreted matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator, key matrix degrading enzymes involved in cell invasion. Concomitantly, HGF stimulated Akt and extracellular signal-regulated kinase (ERK)1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines. Inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway by the PI3K inhibitor, LY294002, markedly suppressed HGF-stimulated invasion of both CCA cell lines, and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION: These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type. PMID:20135719

  16. Inhibitory effects of tetrandrine on epidermal growth factor-induced invasion and migration in HT29 human colorectal adenocarcinoma cells.

    PubMed

    Horng, Chi-Ting; Yang, Jai-Sing; Chiang, Jo-Hua; Lu, Chi-Cheng; Lee, Chiu-Fang; Chiang, Ni-Na; Chen, Fu-An

    2016-01-01

    Tetrandrine has been shown to reduce cancer cell proliferation and to inhibit metastatic effects in multiple cancer models in vitro and in vivo. However, the effects of tetrandrine on the underlying mechanism of HT29 human colorectal adenocarcinoma cell metastasis remain to be fully elucidated. The aim of the present study was focused on tetrandrine‑treated HT29 cells following epidermal growth factor (EGF) treatment, and Transwell, gelatin zymography, gene expression and immunoblotting assays were performed to investigate metastatic effects in vitro. Tetrandrine was observed to dose‑dependently inhibit EGF‑induced HT29 cell invasion and migration, however, no effect on cell viability occurred following exposure to tetradrine between 0.5 and 2 µM. Tetrandrine treatment inhibited the enzymatic activity of matrix metalloprotease (MMP)‑2 and MMP‑9 in a concentration‑dependent manner. The present study also found a reduction in the mRNA expression levels of MMP‑2 and MMP‑9 in the tetrandrine‑treated HT29 cells. Tetrandrine also suppressed the phosphorylation of EGF receptor (EGFR) and its downstream pathway, including phosphoinositide‑dependent kinase 1, phosphatidylinositol 3‑kinase and phosphorylated AKT, suppressing the gene expression of MMP‑2 and MMP‑9. Furthermore, tetrandrine triggered mitogen‑activated protein kinase signaling through the suppressing the activation of phosphorylated extracellular signal‑regulated protein kinase. These data suggested that targeting EGFR signaling and its downstream molecules contributed to the inhibition of EGF‑induced HT29 cell metastasis caused by tetrandrine, eventually leading to a reduction in the mRNA and gelatinase activities of MMP-2 and MMP-9, respectively. PMID:26648313

  17. Mechano-growth factor induces migration of rat mesenchymal stem cells by altering its mechanical properties and activating ERK pathway

    SciTech Connect

    Wu, Jiamin; Wu, Kewen; Lin, Feng; Luo, Qing; Yang, Li; Shi, Yisong; Song, Guanbin; Sung, Kuo-Li Paul

    2013-11-08

    Highlights: •MGF induced the migration of rat MSC in a concentration-dependent manner. •MGF enhanced the mechanical properties of rMSC in inducing its migration. •MGF activated the ERK 1/2 signaling pathway of rMSC in inducing its migration. •rMSC mechanics may synergy with ERK 1/2 pathway in MGF-induced rMSC migration. -- Abstract: Mechano-growth factor (MGF) generated by cells in response to mechanical stimulation has been identified as a mechano effector molecule, playing a key role in regulating mesenchymal stem cell (MSC) function, including proliferation and migration. However, the mechanism(s) underlying how MGF-induced MSC migration occurs is still unclear. In the present study, MGF motivated migration of rat MSCs (rMSCs) in a concentration-dependent manner and optimal concentration of MGF at 50 ng/mL (defined as MGF treatment in this paper) was demonstrated. Notably, enhancement of mechanical properties that is pertinent to cell migration, such as cell traction force and cell stiffness were found to respond to MGF treatment. Furthermore, MGF increased phosphorylation of extracellular signal-regulated kinase (ERK), ERK inhibitor (i.e., PD98059) suppressed ERK phosphorylation, and abolished MGF-induced rMSC migration were found, demonstrating that ERK is involved molecule for MGF-induced rMSC migration. These in vitro evidences of MGF-induced rMSC migration and its direct link to altering rMSC mechanics and activating the ERK pathway, uncover the underlying biomechanical and biological mechanisms of MGF-induced rMSC migration, which may help find MGF-based application of MSC in clinical therapeutics.

  18. Effect of Testosterone on Neuronal Morphology and Neuritic Growth of Fetal Lamb Hypothalamus-Preoptic Area and Cerebral Cortex in Primary Culture.

    PubMed

    Reddy, Radhika C; Amodei, Rebecka; Estill, Charles T; Stormshak, Fred; Meaker, Mary; Roselli, Charles E

    2015-01-01

    Testosterone plays an essential role in sexual differentiation of the male sheep brain. The ovine sexually dimorphic nucleus (oSDN), is 2 to 3 times larger in males than in females, and this sex difference is under the control of testosterone. The effect of testosterone on oSDN volume may result from enhanced expansion of soma areas and/or dendritic fields. To test this hypothesis, cells derived from the hypothalamus-preoptic area (HPOA) and cerebral cortex (CTX) of lamb fetuses were grown in primary culture to examine the direct morphological effects of testosterone on these cellular components. We found that within two days of plating, neurons derived from both the HPOA and CTX extend neuritic processes and express androgen receptors and aromatase immunoreactivity. Both treated and control neurites continue to grow and branch with increasing time in culture. Treatment with testosterone (10 nM) for 3 days significantly (P < 0.05) increased both total neurite outgrowth (35%) and soma size (8%) in the HPOA and outgrowth (21%) and number of branch points (33%) in the CTX. These findings indicate that testosterone-induced somal enlargement and neurite outgrowth in fetal lamb neurons may contribute to the development of a fully masculine sheep brain. PMID:26053052

  19. The transcription of the human fructose-bisphosphate aldolase C gene is activated by nerve-growth-factor-induced B factor in human neuroblastoma cells.

    PubMed Central

    Buono, P; Conciliis, L D; Izzo, P; Salvatore, F

    1997-01-01

    A DNA region located at around -200 bp in the 5' flanking region (region D) of the human brain-type fructose-bisphosphate aldolase (aldolase C) gene has been analysed. We show by transient transfection assay and electrophoretic-mobility-shift assay (EMSA) that the binding of transcriptional activators to region D is much more efficient (80% versus 30%) in human neuroblastoma cells (SKNBE) than in the non-neuronal cell line A1251, which contains low levels of aldolase C mRNA. The sequence of region D, CAAGGTCA, is very similar to the AAAGGTCA motif present in the mouse steroid 21-hydroxylase gene; the latter motif binds nerve-growth-factor-induced B factor (NGFI-B), which is a member of the thyroid/steroid/retinoid nuclear receptor gene family. Competition experiments in EMSA and antibody-directed supershift experiments showed that NGFI-B is involved in the binding to region D of the human aldolase C gene. Furthermore, the regulation of the aldolase C gene (which is the second known target of NGFI-B) expression during development parallels that of NGFI-B. PMID:9173889

  20. Fibroblast growth factor-inducible 14 (Fn14) is expressed in the lower genital tract and may play a role in amplifying inflammation during infection.

    PubMed

    Han, Eugene S; Mekasha, Samrawit; Ingalls, Robin R

    2010-01-01

    TNF-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) cytokine superfamily which regulates a number of cellular responses, including inflammation and proliferation. TWEAK is primarily secreted by phagocytic cells and its receptor, fibroblast growth factor-inducible 14 (Fn14), is expressed on non-lymphoid cells, including epithelial, endothelial and mesenchymal cells. The TWEAK/Fn14 pathway is highly conserved from an evolutionary standpoint, and has been shown to play a role in tissue regeneration and inflammation in the liver, kidney, lung and skeletal muscle. We hypothesized that TWEAK/Fn14 might have a physiological role in regulating infection-induced inflammation in the lower female genital tract. To test this hypothesis, we examined expression of the receptor Fn14 in relevant cells and tissue. Receptor function was tested by treating cells with recombinant TWEAK, with and without other known proinflammatory stimuli. Flow cytometric analysis of vaginal and cervical epithelial cells revealed that Fn14 was highly expressed at the cell surface. We also detected both Fn14 and TWEAK in whole cervical tissue by RT-PCR. Treatment of vaginal and cervical epithelial cells with recombinant TWEAK led to a weak induction of the chemokine IL-8. However, TWEAK potentiated the effects of IL-1ss, the TLR2 ligand Pam(3)CysSK(4), and live Neisseria gonorrhoeae in a synergistic manner. These data reveal a novel pathway for regulation of microbial-induced inflammation in the female reproductive tract and suggest that interference with the TWEAK/Fn14 pathway might be an approach to abrogate excessive infection-induced inflammation caused by sexually transmitted pathogens. PMID:19963275

  1. Tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 are expressed in urticarial vasculitis.

    PubMed

    Li, Mengmeng; Chen, Tao; Guo, Zaipei; Li, Jingyi; Cao, Na

    2013-11-01

    Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, has been implicated as a pro-inflammatory cytokine in many types of autoimmune and infectious diseases. However, information about TWEAK in dermatological diseases is limited. To date, no studies have investigated the roles of TWEAK in patients with urticarial vasculitis (UV). This study aimed to assess serum TWEAK levels, together with TWEAK and fibroblast growth factor-inducible 14 (Fn14) expressions of skin lesions in patients with UV. Serum TWEAK levels in patients with UV, together with patients with cutaneous leukocytoclastic angiitis (CLA) and healthy controls were detected by enzyme-linked immunosorbent assay; TWEAK and Fn14 expressions of skin lesions were analyzed by immunohistochemistry. Results showed that TWEAK and Fn14 were abundantly expressed in the dermal vessel wall of lesional skin in patients with UV but not healthy controls. Serum TWEAK levels in the acute stage in patients with UV were significantly higher than those in the convalescent stage and healthy controls. Serum TWEAK levels were elevated significantly in patients with CLA compared with those in healthy controls. Our previous study indicated that TWEAK may be an important mediator for the development of vascular inflammation in skin. In addition, we also found that TWEAK blockade substantially reduced vascular damage and perivascular leukocyte infiltrates in lipopolysaccharide-induced cutaneous vasculitis. Our study shows that TWEAK may be associated with the pathogenesis of UV; it is therefore suggested that TWEAK may be a potential therapeutic target for UV and other types of cutaneous vasculitis. PMID:23968277

  2. MicroRNA-145 regulates platelet-derived growth factor-induced human aortic vascular smooth muscle cell proliferation and migration by targeting CD40

    PubMed Central

    Li, Yumei; Huang, Jiangnan; Jiang, Zhiyuan; Zhong, Yuanli; Xia, Mingjie; Wang, Hui; Jiao, Yang

    2016-01-01

    The objective of this study is to investigate the expression of microRNA (miR)-145 in human aortic vascular smooth muscle cells (VSMCs) and the effect of miR-145 in the biological behavior and expression of CD40 in VSMCs. Cells were treated with either miR-145 or miR-145 inhibitor. Cell proliferation was analyzed by a colony formation assay and a methyl thiazolyl tetrazolium assay. Cell migration and invasion were assessed using a transwell assay, an invasion assay, and a wound healing assay. A luciferase reporter assay was used to detect the interaction between miR-145 and CD40. Expression of α-SMA, calponin, osteopontin (OPN), epiregulin, activator protein-1 (AP-1) and CD40 was measured using real-time RT-PCR for mRNA levels and Western blotting for protein levels. Overexpression of miR-145 significantly inhibited VSMC proliferation, invasion and migration. Furthermore, OPN, epiregulin, AP-1 and CD40 expression at the mRNA and protein levels was down-regulated by overexpression of miR-145. However, α-SMA and calponin expression at the mRNA and protein levels was up-regulated by overexpression of miR-145. In addition, the luciferase reporter assay showed that CD40 may be a direct target gene of miR-145 in VSMC initiation and development. Moreover, these data demonstrate that the up-regulation of CD40 is critical for miR-145-mediated inhibitory effects on platelet-derived growth factor-induced cell proliferation and migration in human VSMCs. In summary, CD40, a direct target of miR-145, reverses the inhibitory effects of miR-145. These results suggest that the specific modulation of miR-145 in human VSMCs may be an attractive approach for the treatment of proliferative vascular diseases. PMID:27186305

  3. Structural Basis and Targeting of the Interaction between Fibroblast Growth Factor-inducible 14 and Tumor Necrosis Factor-like Weak Inducer of Apoptosis*

    PubMed Central

    Dhruv, Harshil; Loftus, Joseph C.; Narang, Pooja; Petit, Joachim L.; Fameree, Maureen; Burton, Julien; Tchegho, Giresse; Chow, Donald; Yin, Holly; Al-Abed, Yousef; Berens, Michael E.; Tran, Nhan L.; Meurice, Nathalie

    2013-01-01

    Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr176, but not Trp231, resulted in the loss of TWEAK binding to Fn14 substantiating Tyr176 as the anchoring residue. Importantly, mutation of TWEAK at Tyr176 did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers. PMID:24056367

  4. Human central nervous system myelin inhibits neurite outgrowth.

    PubMed

    Ng, W P; Cartel, N; Roder, J; Roach, A; Lozano, A

    1996-05-13

    In vitro and animal studies have identified molecules in mammalian CNS myelin which inhibit neuritic extension and which may be responsible, at least in part, for the lack of axonal regeneration after injury in the injured brain, optic nerve and spinal cord. To determine whether such inhibitory activity may be present in human CNS myelin, we used a bioassay to characterize neurite outgrowth on this substrate. Human CNS myelin strongly inhibited neuritic outgrowth from newborn rat dorsal root ganglion neurons and NG-108-15 cells, a neuroblastoma-glioma hybrid cell line. Similar but less potent inhibitory activity was identified in human gray matter. The CNS myelin inhibition of neuritic outgrowth appeared to be dependent on direct contact between the myelin substrate and neurites. The inhibitory activity in human CNS myelin closely resembled that described in adult rodents. Inhibition of neurite growth by human CNS myelin in this in vitro bioassay mirrors the lack of regeneration in vivo and can be used as a model to develop strategies designed to enhance axonal regeneration and neural recovery. PMID:8782892

  5. Optimizing neurotrophic factor combinations for neurite outgrowth

    NASA Astrophysics Data System (ADS)

    Deister, C.; Schmidt, C. E.

    2006-06-01

    Most neurotrophic factors are members of one of three families: the neurotrophins, the glial cell-line derived neurotrophic factor family ligands (GFLs) and the neuropoietic cytokines. Each family activates distinct but overlapping cellular pathways. Several studies have shown additive or synergistic interactions between neurotrophic factors from different families, though generally only a single combination has been studied. Because of possible interactions between the neurotrophic factors, the optimum concentration of a factor in a mixture may differ from the optimum when applied individually. Additionally, the effect of combinations of neurotrophic factors from each of the three families on neurite extension is unclear. This study examines the effects of several combinations of the neurotrophin nerve growth factor (NGF), the GFL glial cell-line derived neurotrophic factor (GDNF) and the neuropoietic cytokine ciliary neurotrophic factor (CNTF) on neurite outgrowth from young rat dorsal root ganglion (DRG) explants. The combination of 50 ng ml-1 NGF and 10 ng ml-1 of each GDNF and CNTF induced the highest level of neurite outgrowth at a 752 ± 53% increase over untreated DRGs and increased the longest neurite length to 2031 ± 97 µm compared to 916 ± 64 µm for untreated DRGs. The optimum concentrations of the three factors applied in combination corresponded to the optimum concentration of each factor when applied individually. These results indicate that the efficacy of future therapies for nerve repair would be enhanced by the controlled release of a combination of neurotrophins, GFLs and neuropoietic cytokines at higher concentrations than used in previous conduit designs.

  6. Expression of tumor necrosis factor-like weak inducer of apoptosis and fibroblast growth factor-inducible 14 in patients with polymyositis and dermatomyositis

    PubMed Central

    2014-01-01

    Introduction The aim of this study was to investigate the expression of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) in patients with polymyositis (PM) and dermatomyositis (DM), and their relation to clinical manifestations. Methods Serum levels of TWEAK were detected in 98 PM/DM patients and 37 healthy controls by using the ELISA method. Total RNA isolated from fresh-frozen muscle tissue samples of 36 PM/DM patients and 10 healthy controls were used for analyzing the mRNA levels of TWEAK and Fn14 by quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunofluorescence staining of TWEAK and Fn14 was conducted on muscle biopsy specimens from 23 PM/DM patients and seven healthy controls. Results Serum levels of TWEAK were significantly decreased in the PM/DM patients compared to those in the healthy controls (P < 0.001), and serum TWEAK levels negatively correlated with serum CD163 levels in PM/DM patients (r = -0.49, P < 0.001). The expression of Fn14 mRNA was significantly increased in the muscle tissue of PM/DM patients than in the muscle tissue of healthy controls (P < 0.01), whereas the expression of TWEAK mRNA in PM/DM patients was not statistically different from that of the healthy controls (P > 0.05). Fn14 mRNA levels in muscle tissue positively correlated with muscle disease activity (r = 0.512, P < 0.01). Patients with oropharyngeal dysphagia had significantly higher Fn14 mRNA levels than patients without oropharyngeal dysphagia (P < 0.05). The results of immunofluorescence staining showed that 19 out of 23 PM/DM patients were TWEAK-positive, and 20 out of 23 PM/DM patients were Fn14-positive. No detectable expressions of TWEAK or Fn14 were observed in the healthy controls. Conclusions TWEAK-Fn14 axis may be involved in the pathogenesis of PM/DM. Further understanding of TWEAK-Fn14 function in PM/DM may help to define

  7. Rho-associated protein kinase modulates neurite extension by regulating microtubule remodeling and vinculin distribution

    PubMed Central

    Chen, Ke’en; Zhang, Wenbin; Chen, Jing; Li, Sumei; Guo, Guoqing

    2013-01-01

    Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distribution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulating Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite outgrowth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased membrane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vinculin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin. PMID:25206623

  8. Material Stiffness Effects on Neurite Alignment to Photopolymerized Micropatterns

    PubMed Central

    2015-01-01

    The ability to direct neurite growth into a close proximity of stimulating elements of a neural prosthesis, such as a retinal or cochlear implant (CI), may enhance device performance and overcome current spatial signal resolution barriers. In this work, spiral ganglion neurons (SGNs), which are the target neurons to be stimulated by CIs, were cultured on photopolymerized micropatterns with varied matrix stiffnesses to determine the effect of rigidity on neurite alignment to physical cues. Micropatterns were generated on methacrylate thin film surfaces in a simple, rapid photopolymerization step by photomasking the prepolymer formulation with parallel line–space gratings. Two methacrylate series, a nonpolar HMA-co-HDDMA series and a polar PEGDMA-co-EGDMA series, with significantly different surface wetting properties were evaluated. Equivalent pattern periodicity was maintained across each methacrylate series based on photomask band spacing, and the feature amplitude was tuned to a depth of 2 μm amplitude for all compositions using the temporal control afforded by the UV curing methodology. The surface morphology was characterized by scanning electron microscopy and white light interferometry. All micropatterned films adsorb similar amounts of laminin from solution, and no significant difference in SGN survival was observed when the substrate compositions were compared. SGN neurite alignment significantly increases with increasing material modulus for both methacrylate series. Interestingly, SGN neurites respond to material stiffness cues that are orders of magnitude higher (GPa) than what is typically ascribed to neural environments (kPa). The ability to understand neurite response to engineered physical cues and mechanical properties such as matrix stiffness will allow the development of advanced biomaterials that direct de novo neurite growth to address the spatial signal resolution limitations of current neural prosthetics. PMID:25211120

  9. Material stiffness effects on neurite alignment to photopolymerized micropatterns.

    PubMed

    Tuft, Bradley W; Zhang, Lichun; Xu, Linjing; Hangartner, Austin; Leigh, Braden; Hansen, Marlan R; Guymon, C Allan

    2014-10-13

    The ability to direct neurite growth into a close proximity of stimulating elements of a neural prosthesis, such as a retinal or cochlear implant (CI), may enhance device performance and overcome current spatial signal resolution barriers. In this work, spiral ganglion neurons (SGNs), which are the target neurons to be stimulated by CIs, were cultured on photopolymerized micropatterns with varied matrix stiffnesses to determine the effect of rigidity on neurite alignment to physical cues. Micropatterns were generated on methacrylate thin film surfaces in a simple, rapid photopolymerization step by photomasking the prepolymer formulation with parallel line-space gratings. Two methacrylate series, a nonpolar HMA-co-HDDMA series and a polar PEGDMA-co-EGDMA series, with significantly different surface wetting properties were evaluated. Equivalent pattern periodicity was maintained across each methacrylate series based on photomask band spacing, and the feature amplitude was tuned to a depth of 2 μm amplitude for all compositions using the temporal control afforded by the UV curing methodology. The surface morphology was characterized by scanning electron microscopy and white light interferometry. All micropatterned films adsorb similar amounts of laminin from solution, and no significant difference in SGN survival was observed when the substrate compositions were compared. SGN neurite alignment significantly increases with increasing material modulus for both methacrylate series. Interestingly, SGN neurites respond to material stiffness cues that are orders of magnitude higher (GPa) than what is typically ascribed to neural environments (kPa). The ability to understand neurite response to engineered physical cues and mechanical properties such as matrix stiffness will allow the development of advanced biomaterials that direct de novo neurite growth to address the spatial signal resolution limitations of current neural prosthetics. PMID:25211120

  10. The influence of ensheathing cells on olfactory receptor cell neurite outgrowth in vitro.

    PubMed

    Kafitz, K W; Greer, C A

    1998-11-30

    We previously reported that laminin substrates increased primary (1 degree) neurite outgrowth from olfactory receptor cells (ORCs) in vitro. To further explore mechanisms underlying the outgrowth of ORC neurites, we have cocultured ORCs with the ensheathing cells (ENSH) from the olfactory nerve. ORCs were plated either: (i) directly on monolayers of ENSH (prepared with minor modifications as reported by Doucette and Devon, or (ii) on coverslips suspended above the ENSH monolayer to investigate diffusible trophic influences of ENSH. In addition, ORCs were cocultured with either olfactory bulb glia (OBG) or hippocampal astrocytes (HG) or grown on either laminin (LN) substrates or poly-L-lysine (PLL) controls. The length of ORC neurites was determined after 48 hr in vitro. Immunocytochemical characterization of the ENSH cultures for p75 nerve growth factor (NGF) receptor and glial fibrillary acidic protein (GFAP) revealed that those cultures contained more than 80% ENSH. In OBG cultures approximately 10% and in HG cultures no cells with ENSH characteristics were found. All cells with ENSH characteristics were also LN-immunoreactive. After 48 hr in culture ORCs had the longest 1 degree neurites when they were cocultured with ENSH. No significant differences in the 1 degree neurite length were found comparing ORCs grown directly on ENSH and ORCs physically separated from ENSH. ORCs cultured on HG and on EHS-LN showed no significant differences in the ORC 1 degree neurite length, but on both substrates the ORC 1 degree neurites were significantly shorter than on ENSH. The length of the ORC secondary neurites did not vary significantly in the different culture conditions. Our results suggest that while LN appears to contribute to ORC neurite extension, additional diffusible factors released from ENSH are likely to be further determinants of neurite outgrowth. Because the OBG and HG cocultures did not influence ORC neurite outgrowth as significantly as did the ENSH, it

  11. HMG-CoA reductase inhibition causes neurite loss by interfering with geranylgeranylpyrophosphate synthesis.

    PubMed

    Schulz, Joachim G; Bösel, Julian; Stoeckel, Magali; Megow, Dirk; Dirnagl, Ulrich; Endres, Matthias

    2004-04-01

    To determine whether neurite outgrowth depends upon the mevalonate pathway, we blocked mevalonate synthesis in nerve growth factor-treated PC12 cells or primary cortical neurones with atorvastatin, a 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and substituted different intermediates of the mevalonate pathway. We show that HMG-CoA reductase inhibition causes a profound reduction of neurite length, neurite loss and ultimatively cell death in undifferentiated and pre-differentiated PC12 cells and also in rat primary cortical neurones. Geranylgeranylpyrophosphate, but not farnesylpyrophosphate, squalene or cholesterol, completely compensated for the lack of mevalonate. Our data indicate that, under HMG-CoA reductase inhibition, geranylgeranylpyrophosphate rather than farnesylpyrophosphate or cholesterol is critical for neurite outgrowth and/or maintenance. Loss of neurites is an early manifestation of various neurodegenerative disorders, and dysfunction of isoprenylation might play a role in their pathogenesis. PMID:15030386

  12. Intracellular calcium and cyclic nucleotide levels modulate neurite guidance by microtopographical substrate features.

    PubMed

    Li, Shufeng; Tuft, Bradley; Xu, Linjing; Polacco, Marc; Clarke, Joseph C; Guymon, C Allan; Hansen, Marlan R

    2016-08-01

    Micro- and nanoscale surface features have emerged as potential tools to direct neurite growth into close proximity with next generation neural prosthesis electrodes. However, the signaling events underlying the ability of growth cones to respond to topographical features remain largely unknown. Accordingly, this study probes the influence of [Ca(2+) ]i and cyclic nucleotide levels on the ability of neurites from spiral ganglion neurons (SGNs) to precisely track topographical micropatterns. Photopolymerization and photomasking were used to generate micropatterned methacrylate polymer substrates. Dissociated SGN cultures were plated on the micropatterned surfaces. Calcium influx and release from internal stores were manipulated by elevating extracellular K(+) , maintenance in calcium-free media, or bath application of various calcium channel blockers. Cyclic nucleotide activity was increased by application of cpt-cAMP or 8-Br-cGMP. Elevation of [Ca(2+) ]i by treatment of cultures with elevated potassium reduced neurite alignment to physical microfeatures. Maintenance of cultures in Ca(2+) -free medium or treatment with the non-selective voltage-gated calcium channel blocker cadmium or L-type Ca(2+) channel blocker nifedipine did not signficantly alter SGN neurite alignment. By contrast, ryanodine or xestospongin C, which block release of internal calcium stores via ryanodine-sensitive channels or inositol-1,4,5-trisphosphate receptors respectively, each significantly decreased neurite alignment. Cpt-cAMP significantly reduced neurite alignment while 8-Br-cGMP significantly enhanced neurite alignment. Manipulation of [Ca(2+) ]i or cAMP levels significantly disrupts neurite guidance while elevation of cGMP levels increases neurite alignment. The results suggest intracellular signaling pathways similar to those recruited by chemotactic cues are involved in neurite guidance by topographical features. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2037

  13. Controlling Neurite Outgrowth with Patterned Substrates

    PubMed Central

    Yang, In Hong; Co, Carlos C.; Ho, Chia-Chi

    2011-01-01

    In vivo, neurons form neurites, one of which develops into the axon while others become dendrites. While this neuritogenesis process is well programmed in vivo, there are limited methods to control the number and location of neurite extension in vitro. Here we report a method to control neuritogenesis by confining neurons in specific regions using cell resistant poly(oligoethyleneglycol methacrylate-co-methacrylic acid (OEGMA-co-MA)) or poly(ethyleneglycol-block-lactic acid) PEG-PLA. Line patterned substrates reduce multiple extension of neurites and stimulate bi-directional neurite budding for PC12 and cortical neurons. PC12 cells on 20 and 30 µm line patterns extended one neurite in each direction along the line pattern while cortical neuron on 20 and 30 µm line patterns extended one or two neurites in each direction along the line pattern. Statistical analysis of neurite lengths revealed that PC12 cells and cortical neurons on line patterns extend longer neurites. The ability to guide formation of neurites on patterned substrates is useful for generating neural networks and promoting neurite elongation. PMID:21484989

  14. Influence of cAMP and protein kinase A on neurite length from spiral ganglion neurons

    PubMed Central

    Xu, Ningyong; Engbers, Jonathan; Khaja, Sobia; Xu, Linjing; Clark, J. Jason; Hansen, Marlan R.

    2011-01-01

    Regrowth of peripheral spiral ganglion neuron (SGN) fibers is a primary objective in efforts to improve cochlear implant outcomes and to potentially reinnervate regenerated hair cells. Cyclic adenosine monophosphate (cAMP) regulates neurite growth and guidance via activation of protein kinase A (PKA) and Exchange Protein directly Activated by Cylic AMP (Epac). Here we explored the effects of cAMP signaling on SGN neurite length in vitro. We find that the cAMP analog, cpt-cAMP, exerts a biphasic effect on neurite length; increasing length at lower concentrations and reducing length at higher concentrations. This biphasic response occurs in cultures plated on laminin, fibronectin, or tenascin C suggesting that it is not substrate dependent. cpt-cAMP also reduces SGN neurite branching. The Epac-specific agonist, 8-pCPT-2’-O-Me-cAMP, does not alter SGN neurite length. Constitutively active PKA isoforms strongly inhibit SGN neurite length similar to higher levels of cAMP. Chronic membrane depolarization activates PKA in SGNs and also inhibits SGN neurite length. However, inhibition of PKA fails to rescue neurite length in depolarized cultures implying that activation of PKA is not necessary for the inhibition of SGN neurite length by chronic depolarization. Expression of constitutively active phosphatidylinositol 3-kinase, but not c-Jun N-terminal kinase, isoforms partially rescues SGN neurite length in the presence of activated PKA. Taken together, these results suggest that activation of cAMP/PKA represents a potential strategy to enhance SGN fiber elongation following deafness; however such therapies will likely require careful titration so as to simultaneously promote rather than inhibit nerve fiber regeneration. PMID:22154930

  15. The cytoskeleton and neurite initiation

    PubMed Central

    Flynn, Kevin C

    2013-01-01

    Neurons begin their life as simple spheres, but can ultimately assume an elaborate morphology with numerous, highly arborized dendrites, and long axons. This is achieved via an astounding developmental progression which is dependent upon regulated assembly and dynamics of the cellular cytoskeleton. As neurites emerge out of the soma, neurons break their spherical symmetry and begin to acquire the morphological features that define their structure and function. Neurons regulate their cytoskeleton to achieve changes in cell shape, velocity, and direction as they migrate, extend neurites, and polarize. Of particular importance, the organization and dynamics of actin and microtubules directs the migration and morphogenesis of neurons. This review focuses on the regulation of intrinsic properties of the actin and microtubule cytoskeletons and how specific cytoskeletal structures and dynamics are associated with the earliest phase of neuronal morphogenesis—neuritogenesis. PMID:24002528

  16. Immobilized laminin concentration gradients on electrospun fiber scaffolds for controlled neurite outgrowth.

    PubMed

    Zander, Nicole E; Beebe, Thomas P

    2014-03-01

    Neuronal process growth is guided by extrinsic environmental cues such as extracellular matrix (ECM) proteins. Recent reports have described that the growth cone extension is superior across gradients of the ECM protein laminin compared to growth across uniformly distributed laminin. In this work, the authors have prepared gradients of laminin on aligned electrospun nanofibers for use as substrates for neuronal growth. The substrates therefore presented both topographical and chemical guidance cues. Step gradients were prepared by the controlled robotic immersion of plasma-treated polycaprolactone fibers reacted with N-hydroxysuccinimide into the protein solution. The gradients were analyzed using x-ray photoelectron spectroscopy and confocal laser scanning microscopy. Gradients with a dynamic range of protein concentrations were successfully generated and neurite outgrowth was evaluated using neuronlike pheochromocytoma cell line 12 (PC12) cells. After 10 days of culture, PC12 neurite lengths varied from 32.7 ± 14.2 μm to 76.3 ± 9.1 μm across the protein concentration gradient. Neurite lengths at the highest concentration end of the gradient were significantly longer than neurite lengths observed for cells cultured on samples with uniform protein coverage. Gradients were prepared both in the fiber direction and transverse to the fiber direction. Neurites preferentially aligned with the fiber direction in both cases indicating that fiber alignment has a more dominant role in controlling neurite orientation, compared to the chemical gradient. PMID:24739010

  17. In vitro neurite guidance effects induced by polylysine pinstripe micropatterns with polylysine background.

    PubMed

    Joo, Sunghoon; Kang, Kyungtae; Nam, Yoonkey

    2015-08-01

    Engineered culture substrates with chemical neurite guidance cues have been used for studying the mechanism of axon pathfinding at cellular level. In this study, we designed a novel poly-l-lysine (PLL) micropattern ("pinstripe micropattern") to investigate how the same biomolecules with slightly different surface concentration can affect in vitro neuronal growth. The pinstripe micropattern was fabricated by stamping PLL on a PLL-coated glass coverslip, which resulted in denser PLL lines and a less-dense PLL background. There were two effects of the substrate on cultured primary hippocampal neuron: neurite initiation and growth cone turning. Although the whole surface was permissive for neurite outgrowth, we observed that the growth direction of neurites had a strong tendency to follow the stamped PLL line patterns with PLL background. However, the micropattern did not affect the spreading of cell body on the substrate. According to these investigations, we concluded that the PLL pinstripe pattern with PLL background, which had the step difference of polylysine concentrations, would be very useful for designing novel cell assays for the investigation of neurite guidance mechanisms, and suggested it as a new design method for controlling the direction of neurite growth on in vitro neural network. PMID:25630479

  18. Retinoic acid receptor beta2 and neurite outgrowth in the adult mouse spinal cord in vitro.

    PubMed

    Corcoran, Jonathan; So, Po-Lin; Barber, Robert D; Vincent, Karen J; Mazarakis, Nicholas D; Mitrophanous, Kyriacos A; Kingsman, Susan M; Maden, Malcolm

    2002-10-01

    Retinoic acid, acting through the nuclear retinoic acid receptor beta2 (RARbeta2), stimulates neurite outgrowth from peripheral nervous system tissue that has the capacity to regenerate neurites, namely, embryonic and adult dorsal root ganglia. Similarly, in central nervous system tissue that can regenerate, namely, embryonic mouse spinal cord, retinoic acid also stimulates neurite outgrowth and RARbeta2 is upregulated. By contrast, in the adult mouse spinal cord, which cannot regenerate, no such upregulation of RARbeta2 by retinoic acid is observed and no neurites are extended in vitro. To test our hypothesis that the upregulation of RARbeta2 is crucial to neurite regeneration, we have transduced adult mouse or rat spinal cord in vitro with a minimal equine infectious anaemia virus vector expressing RARbeta2. After transduction, prolific neurite outgrowth occurs. Outgrowth does not occur when the cord is transduced with a different isoform of RARbeta nor does it occur following treatment with nerve growth factor. These data demonstrate that RARbeta2 is involved in neurite outgrowth, at least in vitro, and that this gene may in the future be of some therapeutic use. PMID:12235288

  19. The neurite-initiating effect of microbial extracellular glycolipids in PC12 cells.

    PubMed

    Isoda, H; Shinmoto, H; Matsumura, M; Nakahara, T

    1999-09-01

    The effects of several kinds of microbial extracellular glycolipids on neurite initiation in PC12 cells were examined. Addition of mannosylerythritol lipid-A (MEL-A), MEL-B, and sophorose lipid (SL) to PC12 cells caused significant neurite outgrowth. Other glycolipids, such as polyol lipid (PL), rhamnose lipid (RL), succinoyl trehalose lipid-A (STL-A) and STL-B caused no neurite-initiation. MEL-A increased acetylcholine esterase (AChE) activity to an extent similar to nerve growth factor (NGF). However, MEL-A induced one or two long neurites from the cell body, while NGF induced many neurites. In addition, MEL-A-induced differentiation was transient, and after 48 h, percentage of cells with neurites started to decrease in contrast to neurons induced by NGF, which occurred in a time-dependent manner. MEL-A could induce neurite outgrowth after treatment of PC12 cells with an anti-NGF receptor antibody that obstructed NGF action. These results indicate that MEL-A and NGF induce differentiation of PC12 cells through different mechanisms. PMID:19003137

  20. Uridine from Pleurotus giganteus and Its Neurite Outgrowth Stimulatory Effects with Underlying Mechanism

    PubMed Central

    Phan, Chia-Wei; David, Pamela; Wong, Kah-Hui; Naidu, Murali; Sabaratnam, Vikineswary

    2015-01-01

    Neurodegenerative diseases are linked to neuronal cell death and impairment of neurite outgrowth. An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated. The chemical constituents of P. giganteus (linoleic acid, oleic acid, cinnamic acid, caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine) were tested for neurite outgrowth activity. Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1±0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells. Uridine which was present in P. giganteus (1.80±0.03 g/100g mushroom extract) increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt). Further, phosphorylation of the mammalian target of rapamycin (mTOR) was also increased. MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells. This study demonstrated that P. giganteus may enhance neurite outgrowth and one of the key bioactive molecules responsible for neurite outgrowth is uridine. PMID:26565787

  1. Uridine from Pleurotus giganteus and Its Neurite Outgrowth Stimulatory Effects with Underlying Mechanism.

    PubMed

    Phan, Chia-Wei; David, Pamela; Wong, Kah-Hui; Naidu, Murali; Sabaratnam, Vikineswary

    2015-01-01

    Neurodegenerative diseases are linked to neuronal cell death and impairment of neurite outgrowth. An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated. The chemical constituents of P. giganteus (linoleic acid, oleic acid, cinnamic acid, caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine) were tested for neurite outgrowth activity. Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells. Uridine which was present in P. giganteus (1.80 ± 0.03 g/100g mushroom extract) increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt). Further, phosphorylation of the mammalian target of rapamycin (mTOR) was also increased. MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells. This study demonstrated that P. giganteus may enhance neurite outgrowth and one of the key bioactive molecules responsible for neurite outgrowth is uridine. PMID:26565787

  2. Neurite Outgrowth on Nanofiber Scaffolds with Different Orders, Structures, and Surface Properties

    PubMed Central

    Xie, Jingwei; MacEwan, Matthew R.; Li, Xiaoran; Sakiyama-Elbert, Shelly E.; Xia, Younan

    2009-01-01

    Electrospun nanofibers can be readily assembled into various types of scaffolds for applications in neural tissue engineering. The objective of this study is to examine and understand the unique patterns of neurite outgrowth from primary dorsal root ganglia (DRG) cultured on scaffolds of electrospun nanofibers having different orders, structures, and surface properties. We found that the neurites extended radially outward from the DRG main body without specific directionality when cultured on a nonwoven mat of randomly oriented nanofibers. In contrast, the neurites preferentially extended along the long axis of fiber when cultured on a parallel array of aligned nanofibers. When seeded at the border between regions of aligned and random nanofibers, the same DRG simultaneously expressed aligned and random neurite fields in response to the underlying nanofibers. When cultured on a double-layered scaffold where the nanofibers in each layer were aligned along a different direction, the neurites were found to be dependent on the fiber density in both layers. This bi-axial pattern clearly demonstrates that neurite outgrowth can be influenced by nanofibers in different layers of a scaffold, rather than the topmost layer only. Taken together, these results will provide valuable information pertaining to the design of nanofiber scaffolds for neuroregenerative applications, as well as the effects of topology on neurite outgrowth, growth cone guidance, and axonal regeneration. PMID:19397333

  3. Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor-beta 1 and myofibroblast accumulation.

    PubMed Central

    Xing, Z.; Tremblay, G. M.; Sime, P. J.; Gauldie, J.

    1997-01-01

    We have previously reported that transfer to rat lung of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene leads to high expression of GM-CSF between days 1 and 4 and granulation tissue formation followed by an irreversible fibrotic response starting from day 12 onward. In the current study, we investigated the underlying mechanisms. We found that GM-CSF overexpression did not enhance production of tumor necrosis factor-alpha in a significant manner at any time after GM-CSF gene transfer. However, the content of transforming growth factor-beta 1 in bronchoalveolar lavage fluid was markedly induced at day 4 and appeared to be maximal around day 7 and remained high at day 12. Macrophages purified from bronchoalveolar lavage fluid 7 days after GM-CSF gene transfer spontaneously released significant quantities of transforming growth factor-beta 1 protein in vitro. After peak transforming growth factor-beta 1 production was the emergence of alpha-smooth muscle actin-rich myofibroblasts. Accumulation of these cells was most prominent at day 12 within the granulation tissues and they were still present in fibrotic areas between days 12 and 24 and diminished markedly afterward. Thus, we provide the first in vivo evidence that tumor necrosis factor-alpha may be dissociated from participation in a fibrotic process in the lung and GM-CSF may play a more direct role in pulmonary fibrogenesis at least in part through its capability to induce transforming growth factor-beta 1 in macrophages and the subsequent emergence of myofibroblast phenotypes. This GM-CSF transgene lung model is useful for a stepwise dissection of both cellular and molecular events involved in pulmonary fibrosis. Images Figure 2 Figure 5 Figure 6 PMID:9006322

  4. The heat shock protein 90-binding geldanamycin inhibits cancer cell proliferation, down-regulates oncoproteins, and inhibits epidermal growth factor-induced invasion in thyroid cancer cell lines.

    PubMed

    Park, Jin-Woo; Yeh, Michael W; Wong, Mariwil G; Lobo, Margaret; Hyun, William C; Duh, Quan-Yang; Clark, Orlo H

    2003-07-01

    Heat shock protein 90 (HSP90) serves as a chaperone protein and plays a critical role in tumor cell growth and/or survival. Geldanamycin, a specific inhibitor of HSP90, is cytotoxic to several human cancer cell lines, but its effect in thyroid cancer is unknown. We, therefore, investigated the effect of geldanamycin on cell proliferation, oncoprotein expression, and invasion in human thyroid cancer cell lines. We used six thyroid cancer cell lines: TPC-1 (papillary), FTC-133, FTC-236, FTC-238 (follicular), XTC-1 (Hürthle cell), and ARO (anaplastic). We used the dimethyl-thiazol-diphenyltetrazolium bromide assay, a clonogenic assay, an apoptotic assay, and a Matrigel invasion assay. We evaluated oncoprotein expression using Western blots and flow cytometry. After 6 d of treatment with 50 nM geldanamycin, the percent inhibition of growth was 29.4% in TPC-1, 97.5% in FTC-133, 96.7% in FTC-236, 10.8% in FTC-238, 70.9% in XTC-1, and 45.5% in ARO cell lines. In the FTC-133 cell line, geldanamycin treatment decreased clonogenicity by 21% at a concentration of 50 nM; geldanamycin induced apoptosis and down-regulated c-Raf-1, mutant p53, and epidermal growth factor (EGF) receptor expression; geldanamycin inhibited EGF-stimulated invasion. In conclusion, geldanamycin inhibited cancer cell proliferation, down-regulated oncoproteins, and inhibited EGF-induced invasion in thyroid cancer cell lines. PMID:12843186

  5. Myoblasts and myoblast-conditioned medium attract the earliest spinal neurites from frog embryos.

    PubMed Central

    McCaig, C D

    1986-01-01

    A study was made of the capacity of newly segmented somites, unsegmented mesoderm and medium conditioned by each of these tissues to attract the growth of the earliest spinal neurites from the neural tube of Xenopus laevis in tissue culture. When presented with segmented somitic myoblasts or sheets of skin, spinal neurites grew selectively towards the somitic myoblasts. Neurites were not attracted specifically to somitic myoblasts from their own rostrocaudal level. A variable proportion of myoblasts from unsegmented caudal mesoderm differentiated and elongated in co-culture with neural tube and skin. These myoblasts also attracted neural outgrowths, but only if present in sufficient numbers. An agar slab containing medium conditioned by the presence of segmented myoblasts for 1 day attracted neurite outgrowths. A source of medium conditioned by the presence of undifferentiated, unsegmented myotomal mesoderm alone did not attract neurite outgrowths. Nerve growth factor (NGF) at a range of concentrations in the agar source (500-10,000 ng/ml) did not attract the earliest neurite outgrowths. It is concluded that the earliest skeletal myoblasts from Xenopus laevis embryos may attract neural outgrowths by releasing a soluble factor. Myoblasts may have to develop to the stage of somite segmentation before secretion of such an agent begins. The release of a myoblast-derived factor so early in development may assist directed nerve growth in vivo. Images Plate 1 Plate 2 PMID:3795063

  6. TGF-β1, in association with the increased expression of connective tissue growth factor, induce the hypertrophy of the ligamentum flavum through the p38 MAPK pathway.

    PubMed

    Cao, Yan-Lin; Duan, Yang; Zhu, Li-Xin; Zhan, Ye-Nan; Min, Shao-Xiong; Jin, An-Min

    2016-08-01

    Hypertrophy of the ligamentum flavum (LF) is one of the key pathomechanisms of lumbar spinal stenosis (LSS). Transforming growth factor (TGF)-β1 is abundantly expressed in hypertrophied degenerative LF tissues from LSS. However, the molecular mechanisms underling the association between TGF-β1 and LF hypertrophy have not yet been fully elucidated. In this study, we investigated the important role of the mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of LSS by analyzing the expression of connective tissue growth factor (CTGF) and extracellular matrix (ECM) components (collagen I and collagen III) in TGF-β1-treated LF cells. Cell growth assay revealed that TGF-β1, in association with CTGF, enhanced the the proliferation of LF cells, and we found that TGF-β1 also elevated CTGF expression and subsequently enhanced the mRNA expression of collagen I and collagen III. The increased mRNA expression levels of CTGF, collagen I and collagen III were abolished by p38 inhibitors. Both immunofluorescence imaging and western blot analysis of p38 and p-p38 revealed the increased expression and phosphorylation of p38. Silencing the expression of p38 by siRNA in LF cells decreased the protein expression of p38, p-p38 and CTGF, as well as the mRNA expression of CTGF, collagen I and collagen III. Taken together, our findings indicate that TGF-β1, in association with the increased expression of CTGF, contribute to the homeostasis of the ECM and to the hypertrophy of LF through the p38 MAPK pathway. PMID:27279555

  7. TGF-β1, in association with the increased expression of connective tissue growth factor, induce the hypertrophy of the ligamentum flavum through the p38 MAPK pathway

    PubMed Central

    Cao, Yan-Lin; Duan, Yang; Zhu, Li-Xin; Zhan, Ye-Nan; Min, Shao-Xiong; Jin, An-Min

    2016-01-01

    Hypertrophy of the ligamentum flavum (LF) is one of the key pathomechanisms of lumbar spinal stenosis (LSS). Transforming growth factor (TGF)-β1 is abundantly expressed in hypertrophied degenerative LF tissues from LSS. However, the molecular mechanisms underling the association between TGF-β1 and LF hypertrophy have not yet been fully elucidated. In this study, we investigated the important role of the mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of LSS by analyzing the expression of connective tissue growth factor (CTGF) and extracellular matrix (ECM) components (collagen I and collagen III) in TGF-β1-treated LF cells. Cell growth assay revealed that TGF-β1, in association with CTGF, enhanced the the proliferation of LF cells, and we found that TGF-β1 also elevated CTGF expression and subsequently enhanced the mRNA expression of collagen I and collagen III. The increased mRNA expression levels of CTGF, collagen I and collagen III were abolished by p38 inhibitors. Both immunofluorescence imaging and western blot analysis of p38 and p-p38 revealed the increased expression and phosphorylation of p38. Silencing the expression of p38 by siRNA in LF cells decreased the protein expression of p38, p-p38 and CTGF, as well as the mRNA expression of CTGF, collagen I and collagen III. Taken together, our findings indicate that TGF-β1, in association with the increased expression of CTGF, contribute to the homeostasis of the ECM and to the hypertrophy of LF through the p38 MAPK pathway. PMID:27279555

  8. “Spatial Mapping of the Neurite and Soma Proteomes Reveals a Functional Cdc42/Rac Regulatory Network”

    SciTech Connect

    Pertz, Olivier C.; Wang, Yingchun; Yang, Feng; Wang, Wei; gay, laurie J.; Gritsenko, Marina A.; Clauss, Therese RW; Anderson, David J.; Liu, Tao; Auberry, Kenneth J.; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

    2008-02-12

    Neurite extension and growth cone navigation are guided by extracellular cues that control cytoskeletal rearrangements. However, understanding the complex signaling mechanisms that mediate neuritogenesis has been limited by the inability to biochemically separate the neurite and soma for spatial proteomic and bioinformatic analyses. Here, we apply global proteome profiling in combination with a novel neurite purification methodology for comparative analysis of the soma and neurite proteomes of neuroblastoma cells. The spatial relationship of 4855 proteins were mapped revealing networks of signaling proteins that control integrins, the actin cytoskeleton, and axonal guidance in the extending neurite. Bioinformatics and functional analyses revealed a spatially compartmentalized Rac/Cdc42 signaling network that operates in conjunction with multiple GEFs and GAPs to control neurite formation. Interestingly, RNA interference experiments revealed that the different GEFs and GAPs regulate specialized functions during neurite formation including neurite growth and retraction kinetics, cytoskeletal organization, and cell polarity. Our findings provide insight into the spatial organization of signaling networks that enable neuritogenesis and provide a comprehensive system-wide profile of proteins that mediate this process including those that control Rac and Cdc42 signaling.

  9. The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

    PubMed Central

    Lei, Ming-Juan; Wang, Qi; Li, Xiaolin; Chen, Aimin; Luo, Li; Xie, Yajun; Li, Guan; Luo, Da; Mysore, Kirankumar S.; Wen, Jiangqi; Xie, Zhi-Ping; Staehelin, Christian; Wang, Yan-Zhang

    2015-01-01

    Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection. PMID:25794934

  10. The small GTPase ROP10 of Medicago truncatula is required for both tip growth of root hairs and nod factor-induced root hair deformation.

    PubMed

    Lei, Ming-Juan; Wang, Qi; Li, Xiaolin; Chen, Aimin; Luo, Li; Xie, Yajun; Li, Guan; Luo, Da; Mysore, Kirankumar S; Wen, Jiangqi; Xie, Zhi-Ping; Staehelin, Christian; Wang, Yan-Zhang

    2015-03-01

    Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection. PMID:25794934

  11. BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration

    SciTech Connect

    Hinitt, C.A.M.; Wood, J.; Lee, S.S.; Williams, A.C.; Howarth, J.L.; Glover, C.P.; Uney, J.B.; Hague, A.

    2010-08-01

    Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.

  12. 8-Prenylnaringenin inhibits epidermal growth factor-induced MCF-7 breast cancer cell proliferation by targeting phosphatidylinositol-3-OH kinase activity.

    PubMed

    Brunelli, Elisa; Pinton, Giulia; Chianale, Federica; Graziani, Andrea; Appendino, Giovanni; Moro, Laura

    2009-02-01

    8-Prenylnaringenin (8PN), one of the strongest plant-derived oestrogen receptors (ERs) ligand, has been suggested to have potential cancer chemo-preventive activities and anti-angiogenic properties. Because published data suggest that ERs serve as nodal point that allows interactions between hormones and growth factors mediated pathways, we decided to investigate the effects exerted by 8PN on Epidermal growth factor (EGF)-elicited pathways in breast cancer cells. Here we show that in ER positive MCF-7 cells, 8PN interferes with EGF induced cell proliferation by strongly inhibiting activation of PI(3)K/Akt pathway, without affecting EGFR expression or tyrosine phosphorylation, and exerting a synergistic activation of Erk1/2 phosphorylation. Moreover, we demonstrate that 8PN is a direct inhibitor of PI(3)K activity as it is shown by in vitro experiments with the purified enzyme and by its inability to impair serine phosphorylation of a constitutive active form of Akt. These findings suggest that inhibition of PI(3)K is a novel mechanism which contributes to 8PN activity to inhibit cancer cell survival and EGF induced proliferation. PMID:19103290

  13. Growth factor-induced activation of a kinase activity which causes regulatory phosphorylation of p42/microtubule-associated protein kinase.

    PubMed Central

    L'Allemain, G; Her, J H; Wu, J; Sturgill, T W; Weber, M J

    1992-01-01

    p42/microtubule-associated protein kinase (p42mapk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42mapk on the regulatory tyrosine and to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42mapk. This p42mapk-activating activity is capable of phosphorylating a kinase-defective p42mapk mutant, thus confirming its activity as a kinase. Images PMID:1314951

  14. Hepatocyte growth factor-induced up-regulation of Twist drives epithelial-mesenchymal transition in a canine mammary tumour cell line.

    PubMed

    Yoshida, Kota; Choisunirachon, Nan; Saito, Tomochika; Matsumoto, Kaori; Saeki, Kohei; Mochizuki, Manabu; Nishimura, Ryohei; Sasaki, Nobuo; Nakagawa, Takayuki

    2014-12-01

    Epithelial-mesenchymal transition (EMT) is a crucial step in tumour progression. However, the molecular mechanisms underlying EMT in canine tumours remain to be elucidated. In this study, the similarity or difference in the molecular mechanism of EMT in canine cells was evaluated and compared with that reported in human and mouse cells. We used eight cell lines derived from canine mammary cancers. Stimulation with hepatocyte growth factor (HGF) increased cell motility and changed EMT-related markers towards mesenchyme in CHMm cell line. These changes were accompanied by an increase in Twist expression and did not occur in CHMm transfected with Twist siRNA, indicating that Twist plays a key role in this phenomenon in CHMm. However, the down-regulation of E-cadherin was not observed by HGF stimulation. Further studies are required to elucidate the difference between human and canine Twist. PMID:25278141

  15. Hepatocyte growth factor-induced differentiation of bone mesenchymal stem cells toward hepatocyte-like cells occurs through nuclear factor-kappa B signaling in vitro.

    PubMed

    Yang, Tongxi; Wang, Yi; Jiang, Shasha; Liu, Xiaoping; Yu, Zhongjie

    2016-09-01

    Hepatocyte growth factor (HGF) is multifaceted cytokine that regulates proliferation, differentiation, morphology, and motility within numerous stem cells. More recently, HGF has been reported to induce the differentiation of bone mesenchymal stem cells (BMSCs) into mature hepatocytes, but the underlying biochemical and molecular signaling is largely unknown. We isolated BMSC from the bone marrow of rats, which were then cultured and exposed to HGF for 15 days. We subsequently assayed these cells for liver functionality and markers, and blocked NF-кB signaling at various stages of the pathway. The present results demonstrate that HGF induces the differentiation of BMSCs toward hepatocyte-like cells through the NF-кB signaling. More specifically, HGF upregulated the translocation of NF-кB to the nucleus. PMID:27249785

  16. Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway.

    PubMed Central

    Logan, S K; Falasca, M; Hu, P; Schlessinger, J

    1997-01-01

    The signaling events which mediate activation of c-Jun N-terminal kinase (JNK) are not yet well characterized. To broaden our understanding of upstream mediators which link extracellular signals to the JNK pathway, we investigated the role of phosphatidylinositol (PI) 3-kinase in epidermal growth factor (EGF)-mediated JNK activation. In this report we demonstrate that a dominant negative form of PI 3-kinase as well as the inhibitor wortmannin blocks EGF-induced JNK activation dramatically. However, wortmannin does not have an effect on JNK activation induced by UV irradiation or osmotic shock. In addition, a membrane-targeted, constitutively active PI 3-kinase (p110beta) was shown to produce in vivo products and to activate JNK, while a kinase-mutated form of this protein showed no activation. On the basis of these experiments, we propose that PI 3-kinase activity plays a role in EGF-induced JNK activation in these cells. PMID:9315636

  17. Intracellular sphingosine kinase 2-derived sphingosine-1-phosphate mediates epidermal growth factor-induced ezrin-radixin-moesin phosphorylation and cancer cell invasion.

    PubMed

    Adada, Mohamad M; Canals, Daniel; Jeong, Nara; Kelkar, Ashwin D; Hernandez-Corbacho, Maria; Pulkoski-Gross, Michael J; Donaldson, Jane C; Hannun, Yusuf A; Obeid, Lina M

    2015-11-01

    The bioactive sphingolipid sphingosine-1-phosphate (S1P) mediates cellular proliferation, mitogenesis, inflammation, and angiogenesis. These biologies are mediated through S1P binding to specific GPCRs [sphingosine-1-phosphate receptor (S1PR)1-5] and some other less well-characterized intracellular targets. Ezrin-radixin-moesin (ERM) proteins, a family of adaptor molecules linking the cortical actin cytoskeleton to the plasma membrane, are emerging as critical regulators of cancer invasion via regulation of cell morphology and motility. Recently, we identified S1P as an acute ERM activator (via phosphorylation) through its action on S1PR2. In this work, we dissect the mechanism of S1P generation downstream of epidermal growth factor (EGF) leading to ERM phosphorylation and cancer invasion. Using pharmacologic inhibitors, small interfering RNA technologies, and genetic approaches, we demonstrate that sphingosine kinase (SK)2, and not SK1, is essential and sufficient in EGF-mediated ERM phosphorylation in HeLa cells. In fact, knocking down SK2 decreased ERM activation 2.5-fold. Furthermore, we provide evidence that SK2 is necessary to mediate EGF-induced invasion. In addition, overexpressing SK2 causes a 2-fold increase in HeLa cell invasion. Surprisingly, and for the first time, we find that this event, although dependent on S1PR2 activation, does not generate and does not require extracellular S1P secretion, therefore introducing a potential novel model of autocrine/intracrine action of S1P that still involves its GPCRs. These results define new mechanistic insights for EGF-mediated invasion and novel actions of SK2, therefore setting the stage for novel targets in the treatment of growth factor-driven malignancies. PMID:26209696

  18. Expression of Ndufb11 encoding the neuronal protein 15.6 during neurite outgrowth and development.

    PubMed

    Gurok, Ulf; Bork, Kaya; Nuber, Ulrike; Spörle, Ralf; Nöhring, Sabine; Horstkorte, Rüdiger

    2007-01-01

    Neurite outgrowth (e.g. axonal or dendrite outgrowth) of neurons is necessary for the development and functioning of the central nervous system. It is well accepted that the differentiation of neurons and neurite outgrowth involve alterations in gene expression. Furthermore, mitochondria play a role in different aspects of neurite outgrowth. Here we show that the expression of Ndufb11, a gene encoding the mitochondrial protein NP15.6 is decreased in the course of neuronal differentiation. NP15.6 is homologous to the bovine protein ESSS, a component of the mitochondrial complex 1. The homologous human NDUFB11 gene is localized to Xp11.3-Xp11.23, a region associated with neurogenetic disorders. The down-regulation of NP15.6 correlates with neurite outgrowth of PC12 cells induced by nerve growth factor. Furthermore, we analyzed the expression of Ndufb11 in the embryonic and adult mouse. PMID:16962385

  19. Sonic hedgehog stimulates neurite outgrowth in a mechanical stretch model of reactive-astrogliosis

    PubMed Central

    Berretta, Antonio; Gowing, Emma K.; Jasoni, Christine L.; Clarkson, Andrew N.

    2016-01-01

    Although recovery following a stroke is limited, undamaged neurons under the right conditions can establish new connections and take on-board lost functions. Sonic hedgehog (Shh) signaling is integral for developmental axon growth, but its role after injury has not been fully examined. To investigate the effects of Shh on neuronal sprouting after injury, we used an in vitro model of glial scar, whereby cortical astrocytes were mechanically traumatized to mimic reactive astrogliosis observed after stroke. This mechanical trauma impaired neurite outgrowth from post-natal cortical neurons plated on top of reactive astrocytes. Addition of Shh to the media, however, resulted in a concentration-dependent increase in neurite outgrowth. This response was inhibited by cyclopamine and activated by oxysterol 20(S)-hydroxycholesterol, both of which modulate the activity of the Shh co-receptor Smoothened (Smo), demonstrating that Shh-mediated neurite outgrowth is Smo-dependent. In addition, neurite outgrowth was not associated with an increase in Gli-1 transcription, but could be inhibited by PP2, a selective inhibitor of Src family kinases. These results demonstrate that neurons exposed to the neurite growth inhibitory environment associated with a glial scar can be stimulated by Shh, with signaling occurring through a non-canonical pathway, to overcome this suppression and stimulate neurite outgrowth. PMID:26902390

  20. UAP56 is an important mediator of Angiotensin II/platelet derived growth factor induced vascular smooth muscle cell DNA synthesis and proliferation

    SciTech Connect

    Sahni, Abha; Wang, Nadan; Alexis, Jeffrey

    2013-02-15

    Highlights: ► Knockdown of UAP56 inhibits Angiotensin II/PDGF induced vascular smooth muscle cell proliferation. ► UAP56 is a positive regulator of E2F transcriptional activation. ► UAP56 is present in the vessel wall of low flow carotid arteries. -- Abstract: Angiotensin (Ang) II and platelet-derived growth factor (PDGF) are important mediators of pathologic vascular smooth muscle cell (VSMC) proliferation. Identifying downstream mediators of Ang II and PDGF signaling may provide insights for therapies to improve vascular proliferative diseases. We have previously demonstrated that breakpoint cluster region (Bcr) is an important mediator of Ang II/PDGF signaling in VSMC. We have recently reported that the DExD/H box protein UAP56 is an interacting partner of Bcr in regulating VSMC DNA synthesis. We hypothesized that UAP56 itself is an important regulator of VSMC proliferation. In this report we demonstrate that knockdown of UAP56 inhibits Ang II/PDGF induced VSMC DNA synthesis and proliferation, and inhibits E2F transcriptional activity. In addition, we demonstrate that UAP56 is present in the vessel wall of low-flow carotid arteries. These findings suggest that UAP56 is a regulator of VSMC proliferation and identify UAP56 as a target for preventing vascular proliferative disease.

  1. Involvement of cysteine-rich protein 61 in the epidermal growth factor-induced migration of human anaplastic thyroid cancer cells.

    PubMed

    Chin, Li-Han; Hsu, Sung-Po; Zhong, Wen-Bin; Liang, Yu-Chih

    2016-05-01

    Anaplastic thyroid cancer (ATC) is among the most aggressive types of malignant cancer. Epidermal growth factor (EGF) plays a crucial role in the pathogenesis of ATC, and patients with thyroid carcinoma typically exhibit increased cysteine-rich protein 61 (Cyr61). In this study, we found that EGF treatment induced cell migration, stress fiber formation, Cyr61 mRNA and protein expressions, and Cyr61 protein secretion in ATC cells. The recombinant Cyr61 protein significantly induced cell migration; however, inhibition of Cyr61 activity by a Cyr61-specific antibody abrogated EGF-induced cell migration. EGF treatment also affected epithelial-to-mesenchymal transition (EMT)-related marker protein expression, as evidenced by an increase in vimentin and a decrease in E-cadherin expression. Inhibition of Cyr61 expression by Cyr61 siRNA decreased cell migration and reversed the EMT-related marker protein expression. EGF treatment increased the phosphorylation of the extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB), and finally activated Cyr61 promoter plasmid activity. Our results suggest that Cyr61 is induced by EGF through the ERK/CREB signal pathway and that it plays a crucial role in the migration and invasion of ATC cells; moreover, Cyr61 might be a therapeutic target for metastatic ATC. © 2015 Wiley Periodicals, Inc. PMID:25773758

  2. Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells

    PubMed Central

    Hopkins, Mandi M.; Zhang, Zhihong; Liu, Ze; Meier, Kathryn E.

    2016-01-01

    Many key actions of ω-3 (n-3) fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs) in the free fatty acid receptor (FFAR) family, FFA1 (GPR40) and FFA4 (GPR120). n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA), and the tyrosine kinase receptor activated by epidermal growth factor (EGF), was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor. PMID:26821052

  3. Hypoxia- and Vascular Endothelial Growth Factor-Induced Stromal Cell-Derived Factor-1α/CXCR4 Expression in Glioblastomas

    PubMed Central

    Zagzag, David; Esencay, Mine; Mendez, Olga; Yee, Herman; Smirnova, Iva; Huang, Yuanyuan; Chiriboga, Luis; Lukyanov, Eugene; Liu, Mengling; Newcomb, Elizabeth W.

    2008-01-01

    The morphological patterns of glioma cell invasion are known as the secondary structures of Scherer. In this report, we propose a biologically based mechanism for the nonrandom formation of Scherer’s secondary structures based on the differential expression of stromal cell-derived factor (SDF)-1α and CXCR4 at the invading edge of glioblastomas. The chemokine SDF-1α was highly expressed in neurons, blood vessels, subpial regions, and white matter tracts that form the basis of Scherer’s secondary structures. In contrast, the SDF-1α receptor, CXCR4, was highly expressed in invading glioma cells organized around neurons and blood vessels, in subpial regions, and along white matter tracts. Neuronal and endothelial cells exposed to vascular endothelial growth factor up-regulated the expression of SDF-1α. CXCR4-positive tumor cells migrated toward a SDF-1α gradient in vitro, whereas inhibition of CXCR4 expression decreased their migration. Similarly, inhibition of CXCR4 decreased levels of SDF-1α-induced phosphorylation of FAK, AKT, and ERK1/2, suggesting CXCR4 involvement in glioma invasion signaling. These studies offer one plausible molecular basis and explanation of the formation of Scherer’s structures in glioma patients. PMID:18599607

  4. Semi-automatic quantification of neurite fasciculation in high-density neurite images by the Neurite Directional Distribution Analysis (NDDA)

    PubMed Central

    Hopkins, Amy M; Wheeler, Brandon; Staii, Cristian; Kaplan, David L.; Atherton, Timothy J.

    2014-01-01

    Background Bundling of neurite extensions occur during nerve development and regeneration. Understanding the factors that drive neurite bundling is important for designing biomaterials for nerve regeneration toward the innervation target and preventing nociceptive collateral sprouting. High-density neuron cultures including dorsal root ganglia explants are employed for in vitro screening of biomaterials designed to control directional outgrowth. Although some semiautomated image processing methods exist for quantification of neurite outgrowth, methods to quantify axonal fasciculation in terms of direction of neurite outgrowth are lacking. New Method This work presents a semi-automated program to analyze micrographs of high-density neurites; the program aims to quantify axonal fasciculation by determining the orientational distribution function of the tangent vectors of the neurites and calculating its Fourier series coefficients (‘c’ values). Results We found that neurite directional distribution analysis (NDDA) of fasciculated neurites yielded ‘c’ values of ≥ ~0.25 whereas branched outgrowth led to statistically significant lesser values of <~0.2. The ‘c’ values correlated directly to the width of neurite bundles and indirectly to the number of branching points. Comparison with Existing Methods Information about the directional distribution of outgrowth is lost in simple counting methods or achieved laboriously through manual analysis. The NDDA supplements previous quantitative analyses of axonal bundling using a vector-based approach that captures new information about the directionality of outgrowth. Conclusion The NDDA is a valuable addition to open source image processing tools available to biomedical researchers offering a robust, precise approach to quantification of imaged features important in tissue development, disease, and repair. PMID:24680908

  5. Brain-derived neurotrophic factor induces post-lesion transcommissural growth of olivary axons that develop normal climbing fibers on mature Purkinje cells.

    PubMed

    Dixon, Kirsty J; Sherrard, Rachel M

    2006-11-01

    In the adult mammalian central nervous system, reinnervation and recovery from trauma is limited. During development, however, post-lesion plasticity may generate alternate paths providing models to investigate factors that promote reinnervation to appropriate targets. Following unilateral transection of the neonatal rat olivocerebellar pathway, axons from the remaining inferior olive reinnervate the denervated hemicerebellum and develop climbing fiber arbors on Purkinje cells. However, the capacity to recreate this accurate target reinnervation in a mature system remains unknown. In rats lesioned on day 15 (P15) or 30 and treated with intracerebellar injection of brain-derived neurotrophic factor (BDNF) or vehicle 24 h later, the morphology and organisation of transcommissural olivocerebellar reinnervation was examined using neuronal tracing and immunohistochemistry. In all animals BDNF, but not vehicle, induced transcommissural olivocerebellar axonal growth into the denervated hemicerebellum. The distribution of reinnervating climbing fibers was not confined to the injection sites but extended throughout the denervated hemivermis and, less densely, up to 3.5 mm into the hemisphere. Transcommissural olivocerebellar axons were organised into parasagittal microzones that were almost symmetrical to those in the right hemicerebellum. Reinnervating climbing fiber arbors were predominantly normal, but in the P30-lesioned group 10% were either branched within the molecular layer forming a smaller secondary arbor or were less branched, and in the P15 lesion group the reinnervating arbors extended their terminals almost to the pial surface and were larger than control arbors (P < 0.02). These results show that BDNF can induce transcommissural olivocerebellar reinnervation, which resembles developmental neuroplasticity to promote appropriate target reinnervation in a mature environment. PMID:16790241

  6. Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

    PubMed Central

    2013-01-01

    Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis. PMID:23890218

  7. The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1{alpha} expression

    SciTech Connect

    Xu, Wei; Guo, Ting; Zhang, Yan; Jiang, Xiaohong; Zhang, Yongxian; Zen, Ke; Yu, Bo; Zhang, Chen-Yu

    2011-05-01

    Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPAR{gamma}) coactivator-1 alpha (PGC-1{alpha}) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1{alpha} in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1{alpha} expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1{alpha} mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1{alpha} expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1{alpha} by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1{alpha} had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1{alpha} decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPAR{gamma} activation by a PPAR{gamma} antagonist GW9662 abolished the suppressive effects of PGC-1{alpha} on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1{alpha} were enhanced by a PPAR{gamma} agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1{alpha} expression. PGC-1{alpha} suppresses PDGF-induced VSMC migration through PPAR{gamma} coactivation and, consequently, p38 MAPK inhibition.

  8. The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

    SciTech Connect

    Liang Pengfei; Jiang Bimei; Yang Xinghua; Xiao Xianzhong Huang Xu; Long Jianhong; Zhang Pihong; Zhang Minghua; Xiao Muzhang; Xie Tinghong; Huang Xiaoyuan

    2008-10-15

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.

  9. Epidermal growth factor induces Ca(2+) sensitization through Rho-kinase-dependent phosphorylation of myosin phosphatase target subunit 1 in vascular smooth muscle.

    PubMed

    Sasahara, Tomoya; Okamoto, Hiroshi; Ohkura, Natsumi; Kobe, Asami; Yayama, Katsutoshi

    2015-09-01

    We previously found that the protein tyrosine phosphatase inhibitor orthovanadate evoked a vasoconstrictor effect in rat aortas via Rho-kinase-dependent inactivation of myosin light chain phosphatase (MLCP) downstream of epidermal growth factor (EGF) receptor signaling. To determine whether the direct activation of EGF receptor by EGF also induces Rho-kinase-dependent vasoconstriction, isometric tension changes were measured in rat aortic rings without endothelium. Although EGF did not produce a contractile effect, the Ca(2+)-induced force in Ca(2+)-depleted rings significantly increased after treatment with 100nM EGF, suggesting that EGF induces Ca(2+) sensitization by MLCP inactivation. In addition, EGF induced the activation of Rho-kinase and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) in rat aortic smooth muscle cells (VSMCs). The effects of EGF on Ca(2+) sensitivity in aortas and MYPT1 phosphorylation in VSMCs were blocked by inhibitors of EGF receptor (AG1478), Rho-kinase (Y27632), extracellular signal-regulated kinase 1/2 (Erk1/2; FR180204), and mitogen/extracellular signal-regulated kinase (MEK; PD98059), but not by inhibitors of p38 kinase (SB203580) and c-Jun amino-terminal kinase (AS601245). EGF-induced Erk1/2 phosphorylation was not abrogated by the Rho-kinase inhibitor, suggesting that Rho-kinase-dependent phosphorylation of MYPT1 is downstream of EGF receptor/MEK/Erk1/2 signaling. These results suggest that EGF induces Ca(2+) sensitization in vascular smooth muscle by Rho-kinase-dependent inactivation of MLCP mediated by the EGF receptor/MEK/Erk1/2 pathway. PMID:26004531

  10. p70 S6 kinase activation is not required for insulin-like growth factor-induced differentiation of rat, mouse, or human skeletal muscle cells.

    PubMed

    Canicio, J; Gallardo, E; Illa, I; Testar, X; Palacín, M; Zorzano, A; Kaliman, P

    1998-12-01

    Insulin-like growth factors (IGFs) are potent stimulators of muscle differentiation, and phosphatidylinositol 3-kinase (PI 3-kinase) is an essential second messenger in this process. Little is known about the downstream effectors of the IGF/PI 3-kinase myogenic cascade, and contradictory observations have been reported concerning the involvement of p70 S6 kinase. In an attempt to clarify the role of p70 S6 kinase in myogenesis, here we have studied the effect of rapamycin on rat, mouse, and human skeletal muscle cell differentiation. Both insulin and IGF-II activated p70 S6 kinase in rat L6E9 and mouse Sol8 myoblasts, which was markedly inhibited at 1 ng/ml rapamycin concentrations. Consistent with previous observations in a variety of cell lines, rapamycin exerted a potent inhibitory effect on L6E9 and Sol8 serum-induced myoblast proliferation. In contrast, even at high concentrations (20 ng/ml), rapamycin had no effect on IGF-II-induced proliferation or differentiation. Indeed, neither the morphological differentiation, as assessed by myotube formation, nor the expression of muscle-specific markers such as myogenin, myosin heavy chain, or GLUT4 (glucose transporter-4) glucose carriers was altered by rapamycin. Moreover, here we extended our studies on IGF-II-induced myogenesis to human myoblasts derived from skeletal muscle biopsies. We show that, as observed for rat and mouse muscle cells, human myoblasts can be induced to form multinucleated myotubes in the presence of exogenous IGF-II. Moreover, IGF-II-induced human myotube formation was totally blocked by LY294002, a specific PI 3-kinase inhibitor, but remained unaffected in the presence of rapamycin. PMID:9832443

  11. GIT1 enhances neurite outgrowth by stimulating microtubule assembly

    PubMed Central

    Li, Yi-sheng; Qin, Li-xia; Liu, Jie; Xia, Wei-liang; Li, Jian-ping; Shen, Hai-lian; Gao, Wei-Qiang

    2016-01-01

    GIT1, a G-protein-coupled receptor kinase interacting protein, has been reported to be involved in neurite outgrowth. However, the neurobiological functions of the protein remain unclear. In this study, we found that GIT1 was highly expressed in the nervous system, and its expression was maintained throughout all stages of neuritogenesis in the brain. In primary cultured mouse hippocampal neurons from GIT1 knockout mice, there was a significant reduction in total neurite length per neuron, as well as in the average length of axon-like structures, which could not be prevented by nerve growth factor treatment. Overexpression of GIT1 significantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice. The GIT1 N terminal region, including the ADP ribosylation factor-GTPase activating protein domain, the ankyrin domains and the Spa2 homology domain, were sufficient to enhance axonal extension. Importantly, GIT1 bound to many tubulin proteins and microtubule-associated proteins, and it accelerated microtubule assembly in vitro. Collectively, our findings suggest that GIT1 promotes neurite outgrowth, at least partially by stimulating microtubule assembly. This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neurological diseases. PMID:27127481

  12. Ephedrae herba stimulates hepatocyte growth factor-induced MET endocytosis and downregulation via early/late endocytic pathways in gefitinib-resistant human lung cancer cells.

    PubMed

    Nishimura, Yukio; Hyuga, Sumiko; Takiguchi, Soichi; Hyuga, Masashi; Itoh, Kazuyuki; Hanawa, Toshihiko

    2016-05-01

    The MET tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), are known to be overexpressed in a variety of malignant tumor cells, and are implicated in the development of gefitinib-resistance in human non-small cell lung cancer (NSCLC) cells. Ephedrae herba was previously reported to prevent HGF-induced cancer cell motility by directly suppressing HGF/MET signaling through the inhibition of MET tyrosine kinase, and treatment with its extract also considerably reduced MET protein levels. To further investigate the mechanism underlying the Ephedrae herba-induced inhibition of MET phosphorylation as well as its degradation and subsequent disappearance, we examined the effect of Ephedrae herba on HGF-stimulated MET endocytosis and downregulation via early/late endocytic pathways in an NSCLC cell line. Using immunofluorescence microscopy, we found that pretreatment of cells with Ephedrae herba extract dramatically changed the intracellular distribution of plasma membrane-associated MET, and that the resultant MET staining was distributed throughout the cytoplasm. Pretreatment of the cells with Ephedrae herba extract also led to the rapid loss of MET and phosphorylated (p)-MET in HGF-stimulated cells. In contrast, inefficient endocytic delivery of MET and p-MET from early to late endosomes was observed in the absence of Ephedrae herba extract, since considerable amounts of the internalized MET accumulated in the early endosomes and were not delivered to lysosomes up to 1 h after HGF-stimulation. Furthermore, large amounts of MET and p-MET that had accumulated in late endosomes of Ephedrae herba-pretreated cells after HGF stimulation were observed along with bafilomycin A1. Therefore, we inferred that degradation of MET occurred in the late endosome/lysosome pathway. Moreover, western blot analysis revealed the accelerated degradation of MET and p-MET proceeds in cells pretreated with Ephedrae herba extract. Collectively, our results suggest that

  13. Inhibition of aldose reductase prevents growth factor-induced G1-S phase transition through the AKT/phosphoinositide 3-kinase/E2F-1 pathway in human colon cancer cells.

    PubMed

    Ramana, Kota V; Tammali, Ravinder; Srivastava, Satish K

    2010-04-01

    Colon cancer is the leading cause of cancer death in both men and women worldwide. The deregulated cell cycle control or decreased apoptosis of normal epithelial cells leading to uncontrolled proliferation is one of the major features of tumor progression. We have previously shown that aldose reductase (AR), a NADPH-dependent aldo-keto reductase, has been shown to be involved in growth factor-induced proliferation of colon cancer cells. Herein, we report that inhibition of AR prevents epidermal growth factor (EGF)- and basic fibroblast growth factor (bFGF)-induced HT29 cell proliferation by accumulating cells at G(1) phase of cell cycle. Similar results were observed in SW480 and HCT-116 colon cancer cells. Treatment of HT29 cells with AR inhibitor, sorbinil or zopolrestat, prevented the EGF- and bFGF-induced DNA binding activity of E2F-1 and phosphorylation of retinoblastoma protein. Inhibition of AR also prevented EGF- and bFGF-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G(1)-S transition regulatory proteins such as cyclin D1, cdk4, proliferating cell nuclear antigen, cyclin E, and c-myc. More importantly, inhibition of AR prevented the EGF- and bFGF-induced activation of phosphoinositide 3-kinase/AKT and reactive oxygen species generation in colon cancer cells. Further, inhibition of AR also prevented the tumor growth of human colon cancer cells in nude mouse xenografts. Collectively, these results show that AR mediates EGF- and bFGF-induced colon cancer cell proliferation by activating or expressing G(1)-S phase proteins such as E2F-1, cdks, and cyclins through the reactive oxygen species/phosphoinositide 3-kinase/AKT pathway, indicating the use of AR inhibitors in the prevention of colon carcinogenesis. Mol Cancer Ther; 9(4); 813-24. (c)2010 AACR. PMID:20354121

  14. Electrical and Neurotrophin Enhancement of Neurite Outgrowth within a 3D Collagen Scaffold

    PubMed Central

    Adams, Robert D.; Rendell, Sara R.; Counts, Lauren R.; Papke, Jason B.; Willits, Rebecca K.; Harkins, Amy B.

    2016-01-01

    Electrical and chemical stimulation have been studied as potent mechanisms of enhancing nerve regeneration and wound healing. However, it remains unclear how electrical stimuli affect nerve growth, particularly in the presence of neurotrophic factors. The objective of this study was to explore (1) the effect of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) supplementation to support neurite outgrowth in a 3D scaffold, and (2) the effect of brief, low voltage, electrical stimulation (ES) on neurite outgrowth prior to neurotrophin supplementation. Dissociated E11 chick dorsal root ganglia (DRG) were seeded within a 1.5 mg/mL type-I collagen scaffold. For neurotrophin treatments, scaffolds were incubated for 24 hrs in culture media containing nerve growth factor (NGF, 10 ng/mL) or BDNF (200 ng/mL), or both. For ES groups, scaffolds containing neurons were stimulated for 10 min at 8–10 V/m DC, then incubated for 24 hrs with neurotrophin. Fixed and labeled neurons were imaged to measure neurite growth and directionality. BDNF supplementation was not as effective as NGF at supporting DRG neurite outgrowth. ES prior to NGF supplementation improved DRG neurite outgrowth compared to NGF alone. This combination of brief ES with NGF treatment was the most effective treatment compared to NGF or BDNF alone. Brief ES had no impact on neurite directionality in the 3D scaffolds. These results demonstrate that ES improves neurite outgrowth in the presence of neurotrophins, and could provide a potential therapeutic approach to improve nerve regeneration when coupled with neurotrophin treatment. PMID:24710795

  15. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

    PubMed

    Wood, J M; Bold, G; Buchdunger, E; Cozens, R; Ferrari, S; Frei, J; Hofmann, F; Mestan, J; Mett, H; O'Reilly, T; Persohn, E; Rösel, J; Schnell, C; Stover, D; Theuer, A; Towbin, H; Wenger, F; Woods-Cook, K; Menrad, A; Siemeister, G; Schirner, M; Thierauch, K H; Schneider, M R; Drevs, J; Martiny-Baron, G; Totzke, F

    2000-04-15

    PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other

  16. Amyloid β-Protein as a Substrate Interacts with Extracellular Matrix to Promote Neurite Outgrowth

    NASA Astrophysics Data System (ADS)

    Koo, Edward H.; Park, Lisa; Selkoe, Dennis J.

    1993-05-01

    Progressive deposition of amyloid β-protein (Aβ) in brain parenchyma and blood vessels is a characteristic feature of Alzheimer disease. Recent evidence suggests that addition of solubilized synthetic Aβ to medium may produce toxic or trophic effects on cultured hippocampal neurons. Because soluble Aβ may not accumulate in significant quantities in brain, we asked whether immobilized Aβ peptide as a substrate alters neurite outgrowth from cultured rat peripheral sensory neurons. This paradigm may closely mimic the conditions in Alzheimer disease brain tissue, in which neurites contact insoluble, extracellular aggregates of β-amyloid. We detected no detrimental effects of Aβ substrate on neurite outgrowth. Rather, Aβ in combination with low doses of laminin or fibronectin enhanced neurite out-growth from these neuronal explants. Our results suggest that insoluble Aβ in the cerebral neuropil may serve as a neurite-promoting matrix, perhaps explaining the apparent regenerative response of neurites observed around amyloid plaques in Alzheimer disease. Moreover, in concert with the recent discovery of Aβ production by cultured neurons, our data suggest that Aβ plays a normal physiological role in brain by complexing with the extracellular matrix.

  17. Neurite outgrowth and synapse formation by identified leech neurones in culture.

    PubMed

    Chiquet, M; Nicholls, J G

    1987-09-01

    After injury, neurones in the central nervous system (CNS) of the leech regenerate with a high degree of specificity. The aim of our experiments has been to study the sequential steps involved in neurite growth and synapse formation using isolated identified neurones in culture. An important requirement for sprouting of leech neurones is the substrate. Neurites grow only slowly and sparsely on polylysine or vertebrate laminin. The extracellular matrix of leech ganglion capsules contains a protease-sensitive factor which can be extracted with urea. With this material as substrate, growth proceeds rapidly in defined medium. Another neurite-promoting substrate is provided by the plant lectin concanavalin A (Con A). The activity of Con A, but not of the capsule matrix factor, is blocked by the Con A-specific hapten methyl alpha-D-mannoside. The morphology and branching pattern of the neurites in culture depend on the specific substrate and on the type of neurone. During stimulation, less Ca2+ uptake occurs into growth cones than in cell bodies. The mechanism of neurite growth seems not to depend on activity-mediated Ca2+ influx or on interactions between neuronal cell surfaces. However, even without profuse outgrowth, electrical and chemical synapses develop between neighbouring neurones. The type of synapse depends predictably on the types of neurones within the cell pair. Since the development of a synapse can be followed with time in culture, the sequential events can each be studied separately for this multi-step process. PMID:3323399

  18. Differential Intensity-dependent Effects of Magnetic Stimulation on the Longest Neurites and Shorter Dendrites in Neuroscreen-1 Cells

    PubMed Central

    Lin, Ching-Yi; Huang, Whitney J.; Li, Kevin; Swanson, Roy; Cheung, Brian; Lin, Vernon W.; Lee, Yu-Shang

    2015-01-01

    OBJECTIVE Magnetic stimulation (MS) is a potential treatment for neuropsychiatric disorders. This study investigates whether MS-regulated neuronal activity can translate to specific changes in neuronal arborization and thus regulate synaptic activity and function. APPROACH To test our hypotheses, we examined the effects of MS on neurite growth of Neuroscreen-1 (NS-1) cells over pulse frequencies of 1, 5 and 10 Hz at field intensities controlled by machine output (MO). Cells were treated with either 30% or 40% MO and received either maximal or minimal MS-induced current-density. Due to the nature of circular MS coils, the center region of the gridded coverslip (zone 1) received minimal (~5%) electromagnetic current density while the remaining area (zone 2) received maximal (~95%) current density. Plated NS-1 cells were exposed to MS twice per day for 3 days and then evaluated for length and number of neurites and expression of brain-derived neurotrophic factor (BDNF). MAIN RESULTS We show that MS dramatically affects the growth of the longest neurites (axon-like) but does not significantly affect the growth of shorter neurites (dendrite-like). Also, MS-induced changes in the longest neurite growth were most evident in zone 1, but not in zone 2. MS effects were intensity-dependent and were most evident in the bolstering of the longest neurite outgrowth, mainly seen in the 10 Hz MS group. Furthermore, we found that MS-increased BDNF expression and secretion was also frequency-dependent. Taken together, our results show that MS exerts distinct effects when different frequencies and intensities are applied to the neuritic compartments (longest neurite versus shorter dendrite(s)) of NS-1 cells. SIGNIFICANCE These findings support the concept that MS increases BDNF expression and signaling, which sculpts longest neurite arborization and connectivity by which neuronal activity is regulated. Understanding the mechanisms underlying MS is crucial for efficiently incorporating

  19. Differential intensity-dependent effects of magnetic stimulation on the longest neurites and shorter dendrites in neuroscreen-1 cells

    NASA Astrophysics Data System (ADS)

    Lin, Ching-Yi; Huang, Whitney J.; Li, Kevin; Swanson, Roy; Cheung, Brian; Lin, Vernon W.; Lee, Yu-Shang

    2015-04-01

    Objective. Magnetic stimulation (MS) is a potential treatment for neuropsychiatric disorders. This study investigates whether MS-regulated neuronal activity can translate to specific changes in neuronal arborization and thus regulate synaptic activity and function. Approach. To test our hypotheses, we examined the effects of MS on neurite growth of neuroscreen-1 (NS-1) cells over the pulse frequencies of 1, 5 and 10 Hz at field intensities controlled via machine output (MO). Cells were treated with either 30% or 40% MO. Due to the nature of circular MS coils, the center region of the gridded coverslip (zone 1) received minimal (∼5%) electromagnetic current density while the remaining area (zone 2) received maximal (∼95%) current density. Plated NS-1 cells were exposed to MS twice per day for three days and then evaluated for length and number of neurites and expression of brain-derived neurotrophic factor (BDNF). Main results. We show that MS dramatically affects the growth of the longest neurites (axon-like) but does not significantly affect the growth of shorter neurites (dendrite-like). Also, MS-induced changes in the longest neurite growth were most evident in zone 1, but not in zone 2. MS effects were intensity-dependent and were most evident in bolstering longest neurite outgrowth, best seen in the 10 Hz MS group. Furthermore, we found that MS-increased BDNF expression and secretion was also frequency-dependent. Taken together, our results show that MS exerts distinct effects when different frequencies and intensities are applied to the neuritic compartments (longest neurite versus shorter dendrite(s)) of NS-1 cells. Significance. These findings support the concept that MS increases BDNF expression and signaling, which sculpts longest neurite arborization and connectivity by which neuronal activity is regulated. Understanding the mechanisms underlying MS is crucial for efficiently incorporating its use into potential therapeutic strategies.

  20. Stimulation of neuronal neurite outgrowth using functionalized carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Matsumoto, K.; Sato, C.; Naka, Y.; Whitby, R.; Shimizu, N.

    2010-03-01

    Low concentrations (0.11-1.7 µg ml - 1) of functionalized carbon nanotubes (CNTs), which are multi-walled CNTs modified by amino groups, when added with nerve growth factor (NGF), promoted outgrowth of neuronal neurites in dorsal root ganglion (DRG) neurons and rat pheochromocytoma cell line PC12h cells in culture media. The quantity of active extracellular signal-regulated kinase (ERK) was higher after the addition of both 0.85 µg ml - 1 CNTs and NGF than that with NGF alone. CNTs increased the number of cells with neurite outgrowth in DRG neurons and PC12h cells after the inhibition of the ERK signaling pathway using a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Active ERK proteins were detected in MEK inhibitor-treated neurons after the addition of CNTs to the culture medium. These results demonstrate that CNTs may stimulate neurite outgrowth by activation of the ERK signaling pathway. Thus, CNTs are biocompatible and are promising candidates for biological applications and devices.

  1. SOCS3 induces neurite differentiation and promotes neuronal cell survival.

    PubMed

    Mishra, Kanchan Kumar; Gupta, Sakshi; Banerjee, Kakoli

    2016-06-01

    Cytokines and growth factors play an important role in neuronal survival as well as cell death. The family of suppressors of cytokine signalling (SOCS) proteins, which includes SOCS1-7 and cytokine-induced suppressor (CIS), has been shown to act as negative regulators of cytokine-induced signalling. In this report, we highlight the role of SOCS3 in regulating neuronal differentiation and survival. We observed increased SOCS3 expression upon differentiation of PC12 cells as well as neural stem cells. SOCS3 overexpression upregulated differentiation of both neural stem cells and PC12 cells even in the absence of NGF, as evidenced by enhanced neurite outgrowth and upregulation of GAP43, marker associated with neurite outgrowth. siRNA-mediated silencing of SOCS3 confirmed the potential role of SOCS3 in neuritogenesis. We observed that, SOCS3-induced neurite differentiation was mediated via the PI3 kinase pathway. Another interesting observation was that SOCS3 overexpression promoted neuronal cell survival under H2 O2 -mediated stress indicating its fundamental role in cell survival. In conclusion, our results indicate that SOCS3 promotes differentiation and survival of neural cells and could be potentially useful in future therapy for treatment of neurodegenerative disorders. © 2016 IUBMB Life, 68(6):468-476, 2016. PMID:27118613

  2. Immunosuppressant FK506 promotes neurite outgrowth in cultures of PC12 cells and sensory ganglia.

    PubMed Central

    Lyons, W E; George, E B; Dawson, T M; Steiner, J P; Snyder, S H

    1994-01-01

    The immunosuppressant drug FK506 acts by binding to receptor proteins, FK506-binding proteins (FKBPs), which in turn can bind to and regulate a Ca(2+)-dependent phosphatase, calcineurin, and a Ca2+ release channel, the ryanodine receptor. Based on our findings in regeneration models that levels of FKBPs during neural regeneration parallel those of growth-associated protein GAP43, a calcineurin substrate that regulates neurite extension, we examined effects of FK506 in PC12 rat pheochromocytoma cells and in rat sensory ganglia. FK506 enhances neurite outgrowth in both systems by increasing sensitivity to nerve growth factor. Blockade of FK506 actions in sensory ganglia by rapamycin, an FK506 antagonist, establishes that these effects involve FKBPs. Rapamycin itself stimulates neurite outgrowth in PC12 cells. These drug effects are detected at subnanomolar concentrations, suggesting therapeutic application in diseases involving neural degeneration. Images PMID:7512727

  3. Experimental and computational models of neurite extension at a choice point in response to controlled diffusive gradients

    NASA Astrophysics Data System (ADS)

    Catig, G. C.; Figueroa, S.; Moore, M. J.

    2015-08-01

    Ojective. Axons are guided toward desired targets through a series of choice points that they navigate by sensing cues in the cellular environment. A better understanding of how microenvironmental factors influence neurite growth during development can inform strategies to address nerve injury. Therefore, there is a need for biomimetic models to systematically investigate the influence of guidance cues at such choice points. Approach. We ran an adapted in silico biased turning axon growth model under the influence of nerve growth factor (NGF) and compared the results to corresponding in vitro experiments. We examined if growth simulations were predictive of neurite population behavior at a choice point. We used a biphasic micropatterned hydrogel system consisting of an outer cell restrictive mold that enclosed a bifurcated cell permissive region and placed a well near a bifurcating end to allow proteins to diffuse and form a gradient. Experimental diffusion profiles in these constructs were used to validate a diffusion computational model that utilized experimentally measured diffusion coefficients in hydrogels. The computational diffusion model was then used to establish defined soluble gradients within the permissive region of the hydrogels and maintain the profiles in physiological ranges for an extended period of time. Computational diffusion profiles informed the neurite growth model, which was compared with neurite growth experiments in the bifurcating hydrogel constructs. Main results. Results indicated that when applied to the constrained choice point geometry, the biased turning model predicted experimental behavior closely. Results for both simulated and in vitro neurite growth studies showed a significant chemoattractive response toward the bifurcated end containing an NGF gradient compared to the control, though some neurites were found in the end with no NGF gradient. Significance. The integrated model of neurite growth we describe will allow

  4. Ginsenoside-Rd Promotes Neurite Outgrowth of PC12 Cells through MAPK/ERK- and PI3K/AKT-Dependent Pathways.

    PubMed

    Wu, Song-Di; Xia, Feng; Lin, Xue-Mei; Duan, Kang-Li; Wang, Fang; Lu, Qing-Li; Cao, Huan; Qian, Yi-Hua; Shi, Ming

    2016-01-01

    Panax ginseng is a famous herbal medicine widely used in Asia. Ginsenosides have been identified as the principle active ingredients for Panax ginseng's biological activity, among which ginsenoside Rd (Rd) attracts extensive attention for its obvious neuroprotective activities. Here we investigated the effect of Rd on neurite outgrowth, a crucial process associated with neuronal repair. PC12 cells, which respond to nerve growth factor (NGF) and serve as a model for neuronal cells, were treated with different concentrations of Rd, and then their neurite outgrowth was evaluated. Our results showed that 10 μM Rd significantly increased the percentages of long neurite- and branching neurite-bearing cells, compared with respective controls. The length of the longest neurites and the total length of neurites in Rd-treated PC12 cells were much longer than that of respective controls. We also showed that Rd activated ERK1/2 and AKT but not PKC signalings, and inhibition of ERK1/2 by PD98059 or/and AKT by LY294002 effectively attenuated Rd-induced neurite outgrowth. Moreover, Rd upregulated the expression of GAP-43, a neuron-specific protein involved in neurite outgrowth, while PD98059 or/and LY294002 decreased Rd-induced increased GAP-43 expression. Taken together, our results provided the first evidence that Rd may promote the neurite outgrowth of PC12 cells by upregulating GAP-43 expression via ERK- and ARK-dependent signaling pathways. PMID:26840295

  5. Ginsenoside-Rd Promotes Neurite Outgrowth of PC12 Cells through MAPK/ERK- and PI3K/AKT-Dependent Pathways

    PubMed Central

    Wu, Song-Di; Xia, Feng; Lin, Xue-Mei; Duan, Kang-Li; Wang, Fang; Lu, Qing-Li; Cao, Huan; Qian, Yi-Hua; Shi, Ming

    2016-01-01

    Panax ginseng is a famous herbal medicine widely used in Asia. Ginsenosides have been identified as the principle active ingredients for Panax ginseng’s biological activity, among which ginsenoside Rd (Rd) attracts extensive attention for its obvious neuroprotective activities. Here we investigated the effect of Rd on neurite outgrowth, a crucial process associated with neuronal repair. PC12 cells, which respond to nerve growth factor (NGF) and serve as a model for neuronal cells, were treated with different concentrations of Rd, and then their neurite outgrowth was evaluated. Our results showed that 10 μM Rd significantly increased the percentages of long neurite- and branching neurite-bearing cells, compared with respective controls. The length of the longest neurites and the total length of neurites in Rd-treated PC12 cells were much longer than that of respective controls. We also showed that Rd activated ERK1/2 and AKT but not PKC signalings, and inhibition of ERK1/2 by PD98059 or/and AKT by LY294002 effectively attenuated Rd-induced neurite outgrowth. Moreover, Rd upregulated the expression of GAP-43, a neuron-specific protein involved in neurite outgrowth, while PD98059 or/and LY294002 decreased Rd-induced increased GAP-43 expression. Taken together, our results provided the first evidence that Rd may promote the neurite outgrowth of PC12 cells by upregulating GAP-43 expression via ERK- and ARK-dependent signaling pathways. PMID:26840295

  6. Dynamic peripheral traction forces balance stable neurite tension in regenerating Aplysia bag cell neurons

    PubMed Central

    Hyland, Callen; Mertz, Aaron F.; Forscher, Paul; Dufresne, Eric

    2014-01-01

    Growth cones of elongating neurites exert force against the external environment, but little is known about the role of force in outgrowth or its relationship to the mechanical organization of neurons. We used traction force microscopy to examine patterns of force in growth cones of regenerating Aplysia bag cell neurons. We find that traction is highest in the peripheral actin-rich domain and internal stress reaches a plateau near the transition between peripheral and central microtubule-rich domains. Integrating stress over the area of the growth cone reveals that total scalar force increases with area but net tension on the neurite does not. Tensions fall within a limited range while a substantial fraction of the total force can be balanced locally within the growth cone. Although traction continuously redistributes during extension and retraction of the peripheral domain, tension is stable over time, suggesting that tension is a tightly regulated property of the neurite independent of growth cone dynamics. We observe that redistribution of traction in the peripheral domain can reorient the end of the neurite shaft. This suggests a role for off-axis force in growth cone turning and neuronal guidance. PMID:24825441

  7. Dynamic peripheral traction forces balance stable neurite tension in regenerating Aplysia bag cell neurons.

    PubMed

    Hyland, Callen; Mertz, Aaron F; Forscher, Paul; Dufresne, Eric

    2014-01-01

    Growth cones of elongating neurites exert force against the external environment, but little is known about the role of force in outgrowth or its relationship to the mechanical organization of neurons. We used traction force microscopy to examine patterns of force in growth cones of regenerating Aplysia bag cell neurons. We find that traction is highest in the peripheral actin-rich domain and internal stress reaches a plateau near the transition between peripheral and central microtubule-rich domains. Integrating stress over the area of the growth cone reveals that total scalar force increases with area but net tension on the neurite does not. Tensions fall within a limited range while a substantial fraction of the total force can be balanced locally within the growth cone. Although traction continuously redistributes during extension and retraction of the peripheral domain, tension is stable over time, suggesting that tension is a tightly regulated property of the neurite independent of growth cone dynamics. We observe that redistribution of traction in the peripheral domain can reorient the end of the neurite shaft. This suggests a role for off-axis force in growth cone turning and neuronal guidance. PMID:24825441

  8. Facile micropatterning of dual hydrogel systems for 3D models of neurite outgrowth.

    PubMed

    Curley, J Lowry; Moore, Michael J

    2011-12-15

    Understanding how microenvironmental factors influence neurite growth is important to inform studies in nerve regeneration, plasticity, development, and neurophysiology. In vitro models attempting to more accurately mimic the physiological environment by provision of a 3D growth matrix may provide useful foundations. Some limitations of thick 3D culture models include hampered solute transport, less-robust neurite growth than on 2D substrates, and difficulty in achieving spatial control of growth. To this end, we describe a 3D dual hydrogel model for embryonic rat day 15 dorsal root ganglion tissue explant growth using a digital micromirror device for dynamic mask projection photolithography. The photolithography method developed allowed simple, reproducible, one-step fabrication of thick hydrogel constructs on a variety of substrates, including permeable cell culture inserts. The relationships between projected mask size, crosslinked hydrogel resolution, and gel thickness were characterized, and resolution was found generally to decrease with increasing gel thickness. Cell viability in thick (481 μm) hydrogel constructs was significantly greater on permeable supports than glass, suggesting transport limitations were somewhat alleviated. The observed neurite growth was abundant and occurred in a spatially controlled manner throughout the 3D environment, a crucial step in the quest for a more effective biomimetic model of neurite outgrowth. PMID:21936043

  9. Facile micropatterning of dual hydrogel systems for 3D models of neurite outgrowth

    PubMed Central

    Curley, J L; Moore, M J

    2011-01-01

    Understanding how microenvironmental factors influence neurite growth is important to inform studies in nerve regeneration, plasticity, development, and neurophysiology. In vitro models attempting to more accurately mimic the physiological environment by provision of a 3D growth matrix may provide useful foundations. Some limitations of thick 3D culture models include hampered solute transport, less-robust neurite growth than on 2D substrates, and difficulty in achieving spatial control of growth. To this end, we describe a 3D dual hydrogel model for embryonic rat day 15 dorsal root ganglion tissue explant growth using a digital micro-mirror device for dynamic mask projection photolithography. The photolithography method developed allowed simple, reproducible, one-step fabrication of thick hydrogel constructs on a variety of substrates, including permeable cell culture inserts. The relationships between projected mask size, crosslinked hydrogel resolution, and gel thickness were characterized, and resolution was found generally to decrease with increasing gel thickness. Cell viability in thick (481 μm) hydrogel constructs was significantly greater on permeable supports than glass, suggesting transport limitations were somewhat alleviated. The observed neurite growth was abundant and occurred in a spatially controlled manner throughout the 3D environment, a crucial step in the quest for a more effective biomimetic model of neurite outgrowth. PMID:21936043

  10. Identification of molecules in leech extracellular matrix that promote neurite outgrowth.

    PubMed

    Masuda-Nakagawa, L; Beck, K; Chiquet, M

    1988-12-22

    The molecular composition of the substrate is of critical importance for neurite extension by isolated identified leech nerve cells in culture. One substrate upon which rapid growth occurs in defined medium is a cell-free extract of extracellular matrix (ECM) that surrounds the leech central nervous system (CNS). Here we report the co-purification of neurite-promoting activity with a laminin-like molecule. High molecular mass proteins from leech ECM purified by gel filtration exhibited increased specific activity for promoting neurite outgrowth. The most active fractions contained three major polypeptide bands of ca. 340, 250 and 220 kDa. Electron microscopy of rotary-shadowed samples showed three macromolecules, one of which had a cross-shaped structure similar to vertebrate laminin. A second six-armed molecule resembled vertebrate tenascin and a third rod-like molecule resembled vertebrate collagen type IV. The most active fractions contained a protein of ca. 1 MDa on non-reducing gels with disulphide-linked subunits of ca. 220 and 340 kDa, with cross-shaped laminin-like molecules. We conclude that a laminin-like molecule represents a major neurite promoting component present in leech ECM. The experiments represent a first step in determining the location of leech laminin within the CNS and assessing its role in neurite outgrowth during development and regeneration. PMID:2907383

  11. Inhibition of Nischarin Expression Promotes Neurite Outgrowth through Regulation of PAK Activity

    PubMed Central

    Ding, Yuemin; Li, Yuying; Lu, Lingchao; Zhang, Ruyi; Zeng, Linghui; Wang, Linlin; Zhang, Xiong

    2015-01-01

    Nischarin is a cytoplasmic protein expressed in various organs that plays an inhibitory role in cell migration and invasion and the carcinogenesis of breast cancer cells. We previously reported that Nischarin is highly expressed in neuronal cell lines and is differentially expressed in the brain tissue of adult rats. However, the physiological function of Nischarin in neural cells remains unknown. Here, we show that Nischarin is expressed in rat primary cortical neurons but not in astrocytes. Nischarin is localized around the nucleus and dendrites. Using shRNA to knockdown the expression of endogenous Nischarin significantly increases the percentage of neurite-bearing cells, remarkably increases neurite length, and accelerates neurite extension in neuronal cells. Silencing Nischarin expression also promotes dendrite elongation in rat cortical neurons where Nischarin interacts with p21-activated kinase 1/2 (PAK1/2) and negatively regulates phosphorylation of both PAK1 and PAK2. The stimulation of neurite growth observed in cells with decreased levels of Nischarin is partially abolished by IPA3-mediated inhibition of PAK1 activity. Our findings indicate that endogenous Nischarin inhibits neurite outgrowth by blocking PAK1 activation in neurons. PMID:26670864

  12. Essential role of NKCC1 in NGF-induced neurite outgrowth

    SciTech Connect

    Nakajima, Ken-ichi; Miyazaki, Hiroaki; Niisato, Naomi; Marunaka, Yoshinori . E-mail: marunaka@koto.kpu-m.ac.jp

    2007-08-03

    The Na{sup +}/K{sup +}/2Cl{sup -} cotransporter (NKCC) mediates electroneutral transport of 2Cl{sup -} coupled with Na{sup +} and K{sup +} across the plasma membrane, and plays crucial roles in Cl{sup -} uptake into the cells, homeostasis of cellular Cl{sup -}, and cell volume regulation. However, we have very limited information on the roles of ion transporters in neurite outgrowth in neuronal cells. In the present study, we report the role of NKCC1 (an isoform of NKCC) in NGF-induced neurite outgrowth of rat pheochromocytoma PC12D cells. The expression level of NKCC1 protein was increased by NGF treatment. Knock-down of NKCC1 by RNA interference (RNAi) drastically diminished the NGF-induced neurite outgrowth. Transfection of enhanced green fluorescent protein (EGFP)-tagged rat NKCC1 into cells for clarification of intracellular localization of NKCC1 revealed that the EGFP-rNKCC1 was mainly localized in the plasma membrane at growth cone during neurite outgrowth. These observations suggest that NKCC1 plays a fundamental role in NGF-induced neurite outgrowth of PC12D cells.

  13. Pure neuritic leprosy: Current status and relevance.

    PubMed

    Rao, P Narasimha; Suneetha, Sujai

    2016-01-01

    Pure neuritic leprosy has always been an enigma due to its clinical and management ambiguities. Although only the Indian Association of Leprologist's classification recognizes 'pure neuritic leprosy' as a distinct sub group of leprosy, cases nonetheless are reported from various countries of Asia, Africa, South America and Europe, indicating its global relevance. It is important to maintain pure neuritic leprosy as a subgroup as it constitutes a good percentage of leprosy cases reported from India, which contributes to more than half of global leprosy numbers. Unfortunately, a high proportion of these patients present with Grade 2 disability at the time of initial reporting itself due to the early nerve involvement. Although skin lesions are absent by definition, when skin biopsies were performed from the skin along the distribution of the affected nerve, a proportion of patients demonstrated leprosy pathology, revealing sub-clinical skin involvement. In addition on follow-up, skin lesions are noted to develop in up to 20% of pure neuritic leprosy cases, indicating its progression to manifest cutaneous disease. Over the decades, the confirmation of diagnosis of pure neuritic leprosy has been subjective, however, with the arrival and use of high-resolution ultrasonography (HRUS) for nerve imaging, we have a tool not only to objectively measure and record the nerve thickening but also to assess the morphological alterations in the nerve including echo texture, fascicular pattern and vascularity. Management of pure neuritic leprosy requires multidrug therapy along with appropriate dose of systemic corticosteroids, for both acute and silent neuritis. Measures for pain relief, self-care of limbs and physiotherapy are important to prevent as well as manage disabilities in this group of patients. PMID:27088926

  14. MiR-124 is differentially expressed in derivatives of the sympathoadrenal cell lineage and promotes neurite elongation in chromaffin cells.

    PubMed

    Shtukmaster, Stella; Narasimhan, Priyanka; El Faitwri, Tehani; Stubbusch, Jutta; Ernsberger, Uwe; Rohrer, Hermann; Unsicker, Klaus; Huber, Katrin

    2016-08-01

    The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR-124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non-neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growth-factor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal cells. PMID:27094431

  15. Neurite outgrowth enhancement by jiadifenolide: possible targets.

    PubMed

    Shenvi, R A

    2016-04-01

    Covering: 1860-2016A mechanistic link may exist between convulsant plant substances typified by picrotoxinin, and 'neurotrophic' sesquiterpenes like jiadifenolide. Picrotoxinin elicits convulsion by anion blockade of the Cys-loop family of neurotransmitter-gated ion channels. These same receptors mediate neuronal development and neurite outgrowth prior to synapse formation. Due to its structural homology with picrotoxin and anisatin, it is possible that jiadifenolide enhances NGF-stimulated neurite outgrowth by modulation of the Cys-loop family of receptors. PMID:26891462

  16. Intracellular Nogo-A facilitates initiation of neurite formation in mouse midbrain neurons in vitro.

    PubMed

    Kurowska, Z; Brundin, P; Schwab, M E; Li, J-Y

    2014-01-01

    Nogo-A is a transmembrane protein originally discovered in myelin, produced by postnatal CNS oligodendrocytes. Nogo-A induces growth cone collapse and inhibition of axonal growth in the injured adult CNS. In the intact CNS, Nogo-A functions as a negative regulator of growth and plasticity. Nogo-A is also expressed by certain neurons. Neuronal Nogo-A depresses long-term potentiation in the hippocampus and modulates neurite adhesion and fasciculation during development in mice. Here we show that Nogo-A is present in neurons derived from human midbrain (Lund human mesencephalic (LUHMES) cell line), as well as in embryonic and postnatal mouse midbrain (dopaminergic) neurons. In LUHMES cells, Nogo-A was upregulated threefold upon differentiation and neurite extension. Nogo-A was localized intracellularly in differentiated LUHMES cells. Cultured midbrain (dopaminergic) neurons from Nogo-A knock-out mice exhibited decreased numbers of neurites and branches when compared with neurons from wild-type (WT) mice. However, this phenotype was not observed when the cultures from WT mice were treated with an antibody neutralizing plasma membrane Nogo-A. In vivo, neither the regeneration of nigrostriatal tyrosine hydroxylase fibers, nor the survival of nigral dopaminergic neurons after partial 6-hydroxydopamine lesions was affected by Nogo-A deletion. These results indicate that during maturation of cultured midbrain (dopaminergic) neurons, intracellular Nogo-A supports neurite growth initiation and branch formation. PMID:24157929

  17. Fabrication of conductive NGF-conjugated polypyrrole-poly(l-lactic acid) fibers and their effect on neurite outgrowth.

    PubMed

    Zeng, Jingwen; Huang, Zhongbing; Yin, Guangfu; Qin, Jiabang; Chen, Xianchun; Gu, Jianwen

    2013-10-01

    In order to fabricate a tissue scaffold with the neurotrophic and electrical activities, conductive nerve growth factor (NGF)-conjugated polypyrrole-poly(l-lactic acid) (PPy-PLLA) composite fibers were prepared by oxidation polymerization and EDC chemistry with poly-l-lysine. PPy nanoparticles (∼70nm diameter) accumulated on PLLA fiber surface to form a rough thick shell (∼200nm thickness). These NGF-conjugated PPy-PLLA fibers could support PC12 neurite outgrowth and extension. Especially, 40% and 74% increase in PC12 neurite outgrowth and extension, respectively, could be obtained under electrical stimulation of 100mV/cm voltages through the composite fibers. A mechanism for the interaction between neurite extension and the NGF-conjugated PPy-PLLA fibers under electro-stimulation was proposed, to explain the synergistic effect of the rough PPy shell, conjugated NGF and electricity on neurite outgrowth and elongation. PMID:23759386

  18. Nimodipine enhances neurite outgrowth in dopaminergic brain slice co-cultures.

    PubMed

    Sygnecka, Katja; Heine, Claudia; Scherf, Nico; Fasold, Mario; Binder, Hans; Scheller, Christian; Franke, Heike

    2015-02-01

    Calcium ions (Ca(2+)) play important roles in neuroplasticity and the regeneration of nerves. Intracellular Ca(2+) concentrations are regulated by Ca(2+) channels, among them L-type voltage-gated Ca(2+) channels, which are inhibited by dihydropyridines like nimodipine. The purpose of this study was to investigate the effect of nimodipine on neurite growth during development and regeneration. As an appropriate model to study neurite growth, we chose organotypic brain slice co-cultures of the mesocortical dopaminergic projection system, consisting of the ventral tegmental area/substantia nigra and the prefrontal cortex from neonatal rat brains. Quantification of the density of the newly built neurites in the border region (region between the two cultivated slices) of the co-cultures revealed a growth promoting effect of nimodipine at concentrations of 0.1μM and 1μM that was even more pronounced than the effect of the growth factor NGF. This beneficial effect was absent when 10μM nimodipine were applied. Toxicological tests revealed that the application of nimodipine at this higher concentration slightly induced caspase 3 activation in the cortical part of the co-cultures, but did neither affect the amount of lactate dehydrogenase release or propidium iodide uptake nor the ratio of bax/bcl-2. Furthermore, the expression levels of different genes were quantified after nimodipine treatment. The expression of Ca(2+) binding proteins, immediate early genes, glial fibrillary acidic protein, and myelin components did not change significantly after treatment, indicating that the regulation of their expression is not primarily involved in the observed nimodipine mediated neurite growth. In summary, this study revealed for the first time a neurite growth promoting effect of nimodipine in the mesocortical dopaminergic projection system that is highly dependent on the applied concentrations. PMID:25447789

  19. Neurite outgrowth on electrospun PLLA fibers is enhanced by exogenous electrical stimulation

    NASA Astrophysics Data System (ADS)

    Koppes, A. N.; Zaccor, N. W.; Rivet, C. J.; Williams, L. A.; Piselli, J. M.; Gilbert, R. J.; Thompson, D. M.

    2014-08-01

    Objective. Both electrical stimuli (endogenous and exogenous) and topographical cues are instructive to axonal extension. This report, for the first time, investigated the relative dominance of directional topographical guidance cues and directional electrical cues to enhance and/or direct primary neurite extension. We hypothesized the combination of electrical stimulation with electrospun fiber topography would induce longer neurite extension from dorsal root ganglia neurons than the presence of electrical stimulation or aligned topography alone. Approach. To test the hypothesis, neurite outgrowth was examined on laminin-coated poly-L-lactide films or electrospun fibers (2 µm in diameter) in the presence or absence of electrical stimulation. Immunostained neurons were semi-automatically traced using Neurolucida software and morphology was evaluated. Main Results. Neurite extension increased 74% on the aligned fibers compared to film controls. Stimulation alone increased outgrowth by 32% on films or fibers relative to unstimulated film controls. The co-presentation of topographical (fibers) with biophysical (electrical stimulation) cues resulted in a synergistic 126% increase in outgrowth relative to unstimulated film controls. Field polarity had no influence on the directionality of neurites, indicating topographical cues are responsible for guiding neurite extension. Significance. Both cues (electrical stimulation and fiber geometry) are modular in nature and can be synergistically applied in conjunction with other common methods in regenerative medicine such as controlled release of growth factors to further influence axonal growth in vivo. The combined application of electrical and aligned fiber topographical guidance cues described herein, if translated in vivo, could provide a more supportive environment for directed and robust axonal regeneration following peripheral nerve injury.

  20. Neurite Outgrowth On Electrospun PLLA Fibers Is Enhanced By Exogenous Electrical Stimulation

    PubMed Central

    Koppes, A. N.; Zaccor, N. W.; Rivet, C. J.; Williams, L. A.; Piselli, J. M.; Gilbert, R. J.; Thompson, D. M.

    2014-01-01

    Objective Both electrical stimuli (endogenous and exogenous) and topographical cues are instructive to axonal extension. This report, for the first time, investigated the relative dominance of directional topographical guidance cues and directional electrical cues to enhance and/or direct primary neurite extension. We hypothesized the combination of electrical stimulation with electrospun fiber topography would induce longer neurite extension from DRG neurons than the presence of electrical stimulation or aligned topography alone. Approach To test the hypothesis, neurite outgrowth was examined on laminin-coated poly-L-lactide (PLLA) films or electrospun fibers (2 μm in diameter) in the presence or absence of electrical stimulation. Immunostained neurons were semi-automatically traced using Neurolucida software and morphology was evaluated. Results Neurite extension increased 74% on the aligned fibers compared to film controls. Stimulation alone increased outgrowth by 32% on films or fibers relative to unstimulated film controls. The co-presentation of topographical (fibers) with biophysical (electrical stimulation) cues resulted in a synergistic 126% increase in outgrowth relative to unstimulated film controls. Field polarity had no influence on the directionality of neurite, indicating topographical cues are responsible to guide neurite extension. Significance Both cues (electrical stimulation and fiber geometry) are modular in nature and can be synergistically applied in conjunction with other common methods in regenerative medicine such as controlled release of growth factors to further influence axonal growth in vivo. The combined application of electrical and aligned fiber topographical guidance cues described herein, if translated in vivo, could provide a more supportive environment for directed and robust axonal regeneration following peripheral nerve injury. PMID:24891494

  1. Assessment of chemical-induced impairment of human neurite outgrowth by multiparametric live cell imaging in high-density cultures.

    PubMed

    Stiegler, Nina V; Krug, Anne K; Matt, Florian; Leist, Marcel

    2011-05-01

    Chemicals that specifically alter human neurite outgrowth pose a hazard for the development of the nervous system. The identification of such compounds remains a major challenge, especially in a human test system. To address this issue, we developed an imaging-based procedure in LUHMES human neuronal precursor cells to quantify neurite growth of unfixed cultures. Live imaging allowed the simultaneous evaluation of cell viability and neurite outgrowth within one culture dish. The procedure was used to test the hypothesis that inhibitors of specific pathways can impair neurite outgrowth without affecting cell viability. Although the cells were grown at high density to allow extensive networking, overall neurite growth in this complex culture was quantified with a signal-to-noise ratio of > 50. Compounds such as U0126 slowed the extension of neuronal processes at concentrations > 4 times lower than those causing cell death. High numbers of individual viable cells without neurites were identified under such conditions, and neurite outgrowth recovered after washout of the chemical. Also an extension-promoting compound, Y-27632, was identified by this unique multiparametric imaging approach. Finally, the actions of unspecific cytotoxicants such as menadione, cadmium chloride, and sodium dodecyl sulfate were tested to evaluate the specificity of the new assay. We always found a ratio of EC50 (cell death)/EC50 (neurites) < 4 for such chemicals. The described novel test system may thus be useful both for high-throughput screens to identify neuritotoxic agents and for their closer characterization concerning mode of action, compound interactions, or the reversibility of their effects. PMID:21342877

  2. Teneurin-4 promotes cellular protrusion formation and neurite outgrowth through focal adhesion kinase signaling

    PubMed Central

    Suzuki, Nobuharu; Numakawa, Tadahiro; Chou, Joshua; de Vega, Susana; Mizuniwa, Chihiro; Sekimoto, Kaori; Adachi, Naoki; Kunugi, Hiroshi; Arikawa-Hirasawa, Eri; Yamada, Yoshihiko; Akazawa, Chihiro

    2014-01-01

    Teneurin-4 (Ten-4), a transmembrane protein, is highly expressed in the central nervous system; however, its cellular and molecular function in neuronal differentiation remains unknown. In this study, we aimed to elucidate the function of Ten-4 in neurite outgrowth. Ten-4 expression was induced during neurite outgrowth of the neuroblastoma cell line Neuro-2a. Ten-4 protein was localized at the neurite growth cones. Knockdown of Ten-4 expression in Neuro-2a cells decreased the formation of the filopodia-like protrusions and the length of individual neurites. Conversely, overexpression of Ten-4 promoted filopodia-like protrusion formation. In addition, knockdown and overexpression of Ten-4 reduced and elevated the activation of focal adhesion kinase (FAK) and Rho-family small GTPases, Cdc42 and Rac1, key molecules for the membranous protrusion formation downstream of FAK, respectively. Inhibition of the activation of FAK and neural Wiskott-Aldrich syndrome protein (N-WASP), which is a downstream regulator of FAK and Cdc42, blocked protrusion formation by Ten-4 overexpression. Further, Ten-4 colocalized with phosphorylated FAK in the filopodia-like protrusion regions. Together, our findings show that Ten-4 is a novel positive regulator of cellular protrusion formation and neurite outgrowth through the FAK signaling pathway.—Suzuki, N., Numakawa, T., Chou, J., de Vega, S., Mizuniwa, C., Sekimoto, K., Adachi, N., Kunugi, H., Arikawa-Hirasawa, E., Yamada, Y., Akazawa, C. Teneurin-4 promotes cellular protrusion formation and neurite outgrowth through focal adhesion kinase signaling. PMID:24344332

  3. Neurite Outgrowth of Mature Retinal Ganglion Cells and PC12 Cells Requires Activity of CK1δ and CK1ε

    PubMed Central

    Bischof, Joachim; Müller, Adrienne; Fänder, Miriam

    2011-01-01

    Mature retinal ganglion cells (RGCs) do not normally regenerate severed axons after optic nerve injury and show only little neurite outgrowth in culture. However, RGCs can be transformed into an active regenerative state after lens injury (LI) enabling these neurons to regrow axons in vitro and in vivo. In the current study we investigated the role of CK1δ and CK1ε activity in neurite outgrowth of LI stimulated RGCs and nerve growth factor (NGF) stimulated PC12 cells, respectively. In both cell types CK1δ and ε were localized in granular particles aligned at microtubules in neurites and growth cones. Although LI treatment did not measurably affect the expression of CK1δ and ε, it significantly elevated the specific kinase activity in the retina. Similarly, CK1δ/ε specific kinase activity was also elevated in NGF treated PC12 cells compared with untreated controls. Neurite extension in PC12 cells was associated with a change in the activity of CK1δ C-terminal targeting kinases, suggesting that activity of these kinases might be necessary for neurite outgrowth. Pharmacological inactivation of CK1δ and ε markedly compromised neurite outgrowth of both, PC12 cells and LI stimulated RGCs in a concentration dependent manner. These data provide evidence for a so far unknown, but essential role of CK1 isoforms in neurite growth. PMID:21698236

  4. Olanzapine Prevents the PCP-induced Reduction in the Neurite Outgrowth of Prefrontal Cortical Neurons via NRG1.

    PubMed

    Zhang, Qingsheng; Yu, Yinghua; Huang, Xu-Feng

    2016-01-01

    Accumulating evidence suggests that reducing neurite outgrowth and synaptic plasticity plays a critical role in the pathology of cognitive deficits in schizophrenia. The N-methyl-D-aspartate receptor antagonist phencyclidine (PCP) can induce symptoms of schizophrenia as well as reduce dendritic spine density and neurite growth. The antipsychotic drug olanzapine may improve these deficits. This study aimed to investigate: (1) if olanzapine prevents PCP-induced suppression of neurite outgrowth and synaptic protein expression; (2) if olanzapine affects the Akt-GSK3 signaling pathway; and (3) the role of neuregulin 1 (NRG1) in this process. Immunofluorescence revealed that PCP treatment for 24 hours reduces both neurite length (28.5%) and the number of neurite branches (35.6%) in primary prefrontal cortical neuron cultures. PCP reduced protein and mRNA expressions of synaptophysin (24.9% and 23.2%, respectively) and PSD95 (31.5% and 21.4%, respectively), and the protein expression of p-Akt (26.7%) and p-GSK3β (35.2%). Olanzapine co-treatment prevented these PCP-induced effects in normal neurons but not in neurons from NRG1-knockout mice. These results indicate that NRG1 mediates the preventive effects of olanzapine on the PCP-induced impairment of neurite outgrowth and synaptic protein expression. This study provides potential targets for interventions on improving the efficacy of olanzapine on preventing cognitive deficits in schizophrenia. PMID:26781398

  5. Olanzapine Prevents the PCP-induced Reduction in the Neurite Outgrowth of Prefrontal Cortical Neurons via NRG1

    PubMed Central

    Zhang, Qingsheng; Yu, Yinghua; Huang, Xu-Feng

    2016-01-01

    Accumulating evidence suggests that reducing neurite outgrowth and synaptic plasticity plays a critical role in the pathology of cognitive deficits in schizophrenia. The N-methyl-D-aspartate receptor antagonist phencyclidine (PCP) can induce symptoms of schizophrenia as well as reduce dendritic spine density and neurite growth. The antipsychotic drug olanzapine may improve these deficits. This study aimed to investigate: (1) if olanzapine prevents PCP-induced suppression of neurite outgrowth and synaptic protein expression; (2) if olanzapine affects the Akt-GSK3 signaling pathway; and (3) the role of neuregulin 1 (NRG1) in this process. Immunofluorescence revealed that PCP treatment for 24 hours reduces both neurite length (28.5%) and the number of neurite branches (35.6%) in primary prefrontal cortical neuron cultures. PCP reduced protein and mRNA expressions of synaptophysin (24.9% and 23.2%, respectively) and PSD95 (31.5% and 21.4%, respectively), and the protein expression of p-Akt (26.7%) and p-GSK3β (35.2%). Olanzapine co-treatment prevented these PCP-induced effects in normal neurons but not in neurons from NRG1-knockout mice. These results indicate that NRG1 mediates the preventive effects of olanzapine on the PCP-induced impairment of neurite outgrowth and synaptic protein expression. This study provides potential targets for interventions on improving the efficacy of olanzapine on preventing cognitive deficits in schizophrenia. PMID:26781398

  6. Fibroblast Growth Factor Receptor-2 Contributes to the Basic Fibroblast Growth Factor-Induced Neuronal Differentiation in Canine Bone Marrow Stromal Cells via Phosphoinositide 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Nakano, Rei; Edamura, Kazuya; Nakayama, Tomohiro; Narita, Takanori; Okabayashi, Ken; Sugiya, Hiroshi

    2015-01-01

    Bone marrow stromal cells (BMSCs) are considered as candidates for regenerative therapy and a useful model for studying neuronal differentiation. The role of basic fibroblast growth factor (bFGF) in neuronal differentiation has been previously studied; however, the signaling pathway involved in this process remains poorly understood. In this study, we investigated the signaling pathway in the bFGF-induced neuronal differentiation of canine BMSCs. bFGF induced the mRNA expression of the neuron marker, microtubule associated protein-2 (MAP2) and the neuron-like morphological change in canine BMSCs. In the presence of inhibitors of fibroblast growth factor receptors (FGFR), phosphatidylinositol 3-kinase (PI3K) and Akt, i.e., SU5402, LY294002, and MK2206, respectively, bFGF failed to induce the MAP2 mRNA expression and the neuron-like morphological change. bFGF induced Akt phosphorylation, but it was attenuated by the FGFR inhibitor SU5402 and the PI3K inhibitor LY294002. In canine BMSCs, expression of FGFR-1 and FGFR-2 was confirmed, but only FGFR-2 activation was detected by cross-linking and immunoprecipitation analysis. Small interfering RNA-mediated knockdown of FGFR-2 in canine BMSCs resulted in the attenuation of bFGF-induced Akt phosphorylation. These results suggest that the FGFR-2/PI3K/Akt signaling pathway is involved in the bFGF-induced neuronal differentiation of canine BMSCs. PMID:26523832

  7. Scatter factor induces blood vessel formation in vivo.

    PubMed Central

    Grant, D S; Kleinman, H K; Goldberg, I D; Bhargava, M M; Nickoloff, B J; Kinsella, J L; Polverini, P; Rosen, E M

    1993-01-01

    Scatter factor (also known as hepatocyte growth factor) is a glycoprotein secreted by stromal cells that stimulates cell motility and proliferation. In vitro, scatter factor stimulates vascular endothelial cell migration, proliferation, and organization into capillary-like tubes. Using two different in vivo assays, we showed that physiologic quantities of purified native mouse scatter factor and recombinant human hepatocyte growth factor induce angiogenesis (the formation of new blood vessels). The angiogenic activity was blocked by specific anti-scatter factor antibodies. Scatter factor induced cultured microvascular endothelial cells to accumulate and secrete significantly increased quantities of urokinase, an enzyme associated with development of an invasive endothelial phenotype during angiogenesis. We further showed that immunoreactive scatter factor is present surrounding sites of blood vessel formation in psoriatic skin. These findings suggest that scatter factor may act as a paracrine mediator in pathologic angiogenesis associated with human inflammatory disease. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 PMID:7680481

  8. An algorithm for neurite outgrowth reconstruction

    NASA Technical Reports Server (NTRS)

    Weaver, Christina M.; Pinezich, John D.; Lindquist, W. Brent; Vazquez, Marcelo E.

    2003-01-01

    We present a numerical method which provides the ability to analyze digitized microscope images of retinal explants and quantify neurite outgrowth. Few parameters are required as input and limited user interaction is necessary to process an entire experiment of images. This eliminates fatigue related errors and user-related bias common to manual analysis. The method does not rely on stained images and handles images of variable quality. The algorithm is used to determine time and dose dependent, in vitro, neurotoxic effects of 1 GeV per nucleon iron particles in retinal explants. No neurotoxic effects are detected until 72 h after exposure; at 72 h, significant reductions of neurite outgrowth occurred at doses higher than 10 cGy.

  9. Reelin Prevents Apical Neurite Retraction during Terminal Translocation and Dendrite Initiation

    PubMed Central

    O'Dell, Ryan S.; Cameron, David A.; Zipfel, Warren R.

    2015-01-01

    The mechanisms controlling cortical dendrite initiation and targeting are poorly understood. Multiphoton imaging of developing mouse cortex reveals that apical dendrites emerge by direct transformation of the neuron's leading process during the terminal phase of neuronal migration. During this ∼110 min period, the dendritic arbor increases ∼2.5-fold in size and migration arrest occurs below the first stable branch point in the developing arbor. This dendritic outgrowth is triggered at the time of leading process contact with the marginal zone (MZ) and occurs primarily by neurite extension into the extracellular matrix of the MZ. In reeler cortices that lack the secreted glycoprotein Reelin, a subset of neurons completed migration but then retracted and reorganized their arbor in a tangential direction away from the MZ soon after migration arrest. For these reeler neurons, the tangential oriented primary neurites were longer lived than the radially oriented primary neurites, whereas the opposite was true of wild-type (WT) neurons. Application of Reelin protein to reeler cortices destabilized tangential neurites while stabilizing radial neurites and stimulating dendritic growth in the MZ. Therefore, Reelin functions as part of a polarity signaling system that links dendritogenesis in the MZ with cellular positioning and cortical lamination. SIGNIFICANCE STATEMENT Whether the apical dendrite emerges by transformation of the leading process of the migrating neuron or emerges de novo after migration is completed is unclear. Similarly, it is not clear whether the secreted glycoprotein Reelin controls migration and dendritic growth as related or separate processes. Here, multiphoton microscopy reveals the direct transformation of the leading process into the apical dendrite. This transformation is coupled to the successful completion of migration and neuronal soma arrest occurs below the first stable branch point of the nascent dendrite. Deficiency in Reelin causes

  10. Neurite outgrowth resistance to rho kinase inhibitors in PC12 Adh cell.

    PubMed

    Yin, Hua; Hou, Xiaolin; Tao, Tingrui; Lv, Xiaoman; Zhang, Luyong; Duan, Weigang

    2015-05-01

    Rho kinase (ROCK) inhibitor is a promising agent for neural injury disorders, which mechanism is associated with neurite outgrowth. However, neurite outgrowth resistance occurred when PC12 Adh cell was treated with ROCK inhibitors for a longer time. PC12 Adh cells were treated with ROCK inhibitor Y27632 or NGF for different durations. Neurite outgrowth resistance occurred when PC12 Adh cell exposed to Y27632 (33 µM) for 3 or more days, but not happen when exposed to nerve growth factor (NGF, 100 ng/mL). The gene expression in the PC12 Adh cells treated with Y27632 (33 µM) or NGF (100 ng/mL) for 2 or 4 days was assayed by gene microarray, and the reliability of the results were confirmed by real-time RT-PCR. Cluster analysis proved that the gene expression profile of PC12 Adh cell treated with Y27632 for 4 days was different from that treated with Y27632 for 2 days and those treated with NGF for 2 and 4 days, respectively. Pathway analysis hinted that the neurite outgrowth resistance could be associated with up-regulation of inflammatory pathways, especially rno04610 (complement and coagulation cascades), and down-regulation of cell cycle pathways, especially rno04110. PMID:25571866

  11. Versican V1 Isoform Induces Neuronal Differentiation and Promotes Neurite Outgrowth

    PubMed Central

    Wu, Yaojiong; Sheng, Wang; Chen, Liwen; Dong, Haiheng; Lee, Vivian; Lu, Fred; Wong, C. Shun; Lu, Wei-Yang; Yang, Burton B.

    2004-01-01

    The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adult brain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced an aborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failed to do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustained extracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, β1 integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform also promoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versican regulates neuronal development, function, and repair. PMID:14978219

  12. The effects of neurotrophin-3 and brain-derived neurotrophic factor on cerebellar granule cell movement and neurite extension in vitro.

    PubMed

    Tanaka, S; Sekino, Y; Shirao, T

    2000-01-01

    Migration of the granule cells is a major stage of cerebellar maturation. Granule cells express neurotrophins and their receptors; however, their role in cell migration has not been defined. In this study we investigated the effects of exogenous neurotrophins on the movement and neurite extension of granule cells from glial-free cerebellar cell reaggregates in vitro. Our results provide direct evidence that neurotrophin-3 and brain-derived neurotrophic factor differentially affect the granule cells. Neurotrophin-3 significantly affected granule cell movements by decreasing the migration index (the ratio of the number of cells that moved further than half the neurite length) and the speed of cell soma movement, but did not affect neurite length or growth cone migration. In contrast, brain-derived neurotrophic factor and neurotrophin-4 acted on growing neurites and growth cones by significantly increasing neurite length and the speed of growth cone migration, but had no effect either on the migration index or on the speed of the cell soma movement. The results suggest that neurotrophins differentially affect neurite extension and the movements of cerebellar granule cells. PMID:10842017

  13. The combinatorics of neurite self-avoidance.

    PubMed

    Forbes, Elizabeth M; Hunt, Jonathan J; Goodhill, Geoffrey J

    2011-11-01

    During neural development in Drosophila, the ability of neurite branches to recognize whether they are from the same or different neurons depends crucially on the molecule Dscam1. In particular, this recognition depends on the stochastic acquisition of a unique combination of Dscam1 isoforms out of a large set of possible isoforms. To properly interpret these findings, it is crucial to understand the combinatorics involved, which has previously been attempted only using stochastic simulations for some specific parameter combinations. Here we present closed-form solutions for the general case. These reveal the relationships among the key variables and how these constrain possible biological scenarios. PMID:21732864

  14. Zonisamide Enhances Neurite Elongation of Primary Motor Neurons and Facilitates Peripheral Nerve Regeneration In Vitro and in a Mouse Model

    PubMed Central

    Yagi, Hideki; Ohkawara, Bisei; Nakashima, Hiroaki; Ito, Kenyu; Tsushima, Mikito; Ishii, Hisao; Noto, Kimitoshi; Ohta, Kyotaro; Masuda, Akio; Imagama, Shiro; Ishiguro, Naoki; Ohno, Kinji

    2015-01-01

    No clinically applicable drug is currently available to enhance neurite elongation after nerve injury. To identify a clinically applicable drug, we screened pre-approved drugs for neurite elongation in the motor neuron-like NSC34 cells. We found that zonisamide, an anti-epileptic and anti-Parkinson’s disease drug, promoted neurite elongation in cultured primary motor neurons and NSC34 cells in a concentration-dependent manner. The neurite-scratch assay revealed that zonisamide enhanced neurite regeneration. Zonisamide was also protective against oxidative stress-induced cell death of primary motor neurons. Zonisamide induced mRNA expression of nerve growth factors (BDNF, NGF, and neurotrophin-4/5), and their receptors (tropomyosin receptor kinase A and B). In a mouse model of sciatic nerve autograft, intragastric administration of zonisamide for 1 week increased the size of axons distal to the transected site 3.9-fold. Zonisamide also improved the sciatic function index, a marker for motor function of hindlimbs after sciatic nerve autograft, from 6 weeks after surgery. At 8 weeks after surgery, zonisamide was protective against denervation-induced muscle degeneration in tibialis anterior, and increased gene expression of Chrne, Colq, and Rapsn, which are specifically expressed at the neuromuscular junction. We propose that zonisamide is a potential therapeutic agent for peripheral nerve injuries as well as for neuropathies due to other etiologies. PMID:26571146

  15. SCYL1BP1 modulates neurite outgrowth and regeneration by regulating the Mdm2/p53 pathway

    PubMed Central

    Liu, Yonghua; Chen, Ying; Lu, Xiang; Wang, Youhua; Duan, Yinong; Cheng, Chun; Shen, Aiguo

    2012-01-01

    SCY1-like 1–binding protein 1 (SCYL1BP1) is a newly identified transcriptional activator domain containing a protein with many unknown biological functions. Recently emerging evidence has revealed that it is a novel regulator of the p53 pathway, which is required for neurite outgrowth and regeneration. Here we present evidence that SCYL1BP1 inhibits nerve growth factor–mediated neurite outgrowth in PC12 cells and affects morphogenesis of primary cortical neurons by strongly decreasing the p53 protein level in vitro, all of which depends on SCYL1BP1's transcriptional activator domain. Exogenous p53 rescues neurite outgrowth and neuronal morphogenesis defects caused by SCYL1BP1. Furthermore, SCYL1BP1 can directly induce Mdm2 transcription, whereas inhibiting the function of Mdm2 by specific small interfering RNAs results in partial rescue of neurite outgrowth and neuronal morphogenesis defects induced by SCYL1BP1. In vivo experiments show that SCYL1BP1 can also depress axonal regeneration, whereas inhibiting the function of SCYL1BP1 by specific short hairpin RNA enhances it. Taken together, these data strongly suggested that SCYL1BP1 is a novel transcriptional activator in neurite outgrowth by directly modulating the Mdm2/p53-dependent pathway, which might play an important role in CNS development and axonal regeneration after injury. PMID:23051735

  16. Senile plaque neurites in Alzheimer disease accumulate amyloid precursor protein.

    PubMed Central

    Cras, P; Kawai, M; Lowery, D; Gonzalez-DeWhitt, P; Greenberg, B; Perry, G

    1991-01-01

    Senile plaques are polymorphous beta-amyloid protein deposits found in the brain in Alzheimer disease and normal aging. This beta-amyloid protein is derived from a larger precursor molecule of which neurons are the principal producers in brain. We found that amyloid precursor protein (APP)-immunoreactive neurites were involved in senile plaques and that only a subset of these neurites showed markers for the abnormal filaments characteristic of neurofibrillary pathology. In the neocortex of nondemented individuals with senile plaques but spared of neurofibrillary pathology, dystrophic neurites in senile plaques showed only APP accumulation. In contrast, in the brains of Alzheimer patients, virtually all APP-immunoreactive neurites also showed immunoreactivity with ubiquitin, tau, and phosphorylated neurofilaments. The presence of tau and neurofilament epitopes in dystrophic neurites in senile plaques was correlated with the extent of neurofibrillary pathology in the surrounding brain tissue. Accumulation of APP and the formation of neurofibrillary pathology in senile plaque neurites are therefore distinct phenomena. Our findings suggest that APP accumulation in senile plaque neurites occurs prior to tau accumulation and is therefore more closely related to appearance of neuritic dystrophy. Images PMID:1652752

  17. Initial neurite outgrowth in Drosophila neurons is driven by kinesin-powered microtubule sliding

    PubMed Central

    Lu, Wen; Fox, Pangkong; Lakonishok, Margot; Davidson, Michael W.; Gelfand, Vladimir I.

    2013-01-01

    Summary Remarkably, forces within a neuron can extend its axon to a target that could be meters away. The two main cytoskeleton components in neurons are microtubules, which are mostly bundled along the axon shaft, and actin filaments, which are highly enriched in a structure at the axon distal tip, the growth cone. Neurite extension has been thought to be driven by a combination of two forces: pushing via microtubule assembly and/or pulling by an actin-driven mechanism in the growth cone [1, 2]. Here we show that a novel mechanism, sliding of microtubules against each other by the microtubule motor kinesin-1 provides the mechanical forces necessary for initial neurite extension in Drosophila neurons. Neither actin filaments in the growth cone nor tubulin polymerization is required for initial outgrowth. Microtubule sliding in neurons is developmentally regulated and is suppressed during neuronal maturation. As kinesin-1 is highly evolutionarily conserved from Drosophila to humans, it is likely that kinesin-1-powered microtubule sliding plays an important role in neurite extension in many types of neurons across species. PMID:23707427

  18. Graphene substrate for inducing neurite outgrowth.

    PubMed

    Lee, Jeong Soon; Lipatov, Alexey; Ha, Ligyeom; Shekhirev, Mikhail; Andalib, Mohammad Nahid; Sinitskii, Alexander; Lim, Jung Yul

    2015-05-01

    A few recent studies demonstrated that graphene may have cytocompatibility with several cell types. However, when assessing cell behavior on graphene, there has been no precise control over the quality of graphene, number of graphene layers, and substrate surface coverage by graphene. In this study, using well-controlled monolayer graphene film substrates we tested the cytocompatibility of graphene for human neuroblastoma (SH-SY5Y) cell culture. A large-scale monolayer graphene film grown on Cu foils by chemical vapor deposition (CVD) could be successfully transferred onto glass substrates by wet transfer technique. We observed that graphene substrate could induce enhanced neurite outgrowth, both in neurite length and number, compared with control glass substrate. Interestingly, the positive stimulatory effect by graphene was achieved even in the absence of soluble neurogenic factor, retinoic acid (RA). Key genes relevant to cell neurogenesis, e.g., neurofilament light chain (NFL), were also upregulated on graphene. Inhibitor studies suggested that the graphene stimulation of cellular neurogenesis may be achieved through focal adhesion kinase (FAK) and p38 mitogen-activated protein kinase (MAPK) cascades. Our data indicate that graphene may be exploited as a platform for neural regenerative medicine, and the suggested molecular mechanism may provide an insight into the graphene control of neural cells. PMID:25778866

  19. Induction of neurite outgrowth in 3D hydrogel-based environments.

    PubMed

    Assunção-Silva, Rita C; Oliveira, Cátia Costa; Ziv-Polat, Ofra; Gomes, Eduardo D; Sahar, Abraham; Sousa, Nuno; Silva, Nuno A; Salgado, António J

    2015-09-01

    The ability of peripheral nervous system (PNS) axons to regenerate and re-innervate their targets after an injury has been widely recognized. However, despite the considerable advances made in microsurgical techniques, complete functional recovery is rarely achieved, especially for severe peripheral nerve injuries (PNIs). Therefore, alternative therapies that can successfully repair peripheral nerves are still essential. In recent years the use of biodegradable hydrogels enriched with growth-supporting and guidance cues, cell transplantation, and biomolecular therapies have been explored for the treatment of PNIs. Bearing this in mind, the aim of this study was to assess whether Gly-Arg-Gly-Asp-Ser synthetic peptide (GRGDS)-modified gellan gum (GG) based hydrogels could foster an amenable environment for neurite/axonal growth. Additionally, strategies to further improve the rate of neurite outgrowth were also tested, namely the use of adipose tissue derived stem cells (ASCs), as well as the glial derived neurotrophic factor (GDNF). In order to increase its stability and enhance its bioactivity, the GDNF was conjugated covalently to iron oxide nanoparticles (IONPs). The impact of hydrogel modification as well as the effect of the GDNF-IONPs on ASC behavior was also screened. The results revealed that the GRGDS-GG hydrogel was able to support dorsal root ganglia (DRG)-based neurite outgrowth, which was not observed for non-modified hydrogels. Moreover, the modified hydrogels were also able to support ASCs attachment. In contrast, the presence of the GDNF-IONPs had no positive or negative impact on ASC behavior. Further experiments revealed that the presence of ASCs in the hydrogel improved axonal growth. On the other hand, GDNF-IONPs alone or combined with ASCs significantly increased neurite outgrowth from DRGs, suggesting a beneficial role of the proposed strategy for future applications in PNI regenerative medicine. PMID:26480959

  20. Active Achilles tendon kinesitherapy accelerates Achilles tendon repair by promoting neurite regeneration☆

    PubMed Central

    Jielile, Jiasharete; Aibai, Minawa; Sabirhazi, Gulnur; Shawutali, Nuerai; Tangkejie, Wulanbai; Badelhan, Aynaz; Nuerduola, Yeermike; Satewalede, Turde; Buranbai, Darehan; Hunapia, Beicen; Jialihasi, Ayidaer; Bai, Jingping; Kizaibek, Murat

    2012-01-01

    Active Achilles tendon kinesitherapy facilitates the functional recovery of a ruptured Achilles tendon. However, protein expression during the healing process remains a controversial issue. New Zealand rabbits, aged 14 weeks, underwent tenotomy followed immediately by Achilles tendon microsurgery to repair the Achilles tendon rupture. The tendon was then immobilized or subjected to postoperative early motion treatment (kinesitherapy). Mass spectrography results showed that after 14 days of motion treatment, 18 protein spots were differentially expressed, among which, 12 were up-regulated, consisting of gelsolin isoform b and neurite growth-related protein collapsing response mediator protein 2. Western blot analysis showed that gelsolin isoform b was up-regulated at days 7–21 of motion treatment. These findings suggest that active Achilles tendon kinesitherapy promotes the neurite regeneration of a ruptured Achilles tendon and gelsolin isoform b can be used as a biomarker for Achilles tendon healing after kinesitherapy. PMID:25317130

  1. Active Achilles tendon kinesitherapy accelerates Achilles tendon repair by promoting neurite regeneration.

    PubMed

    Jielile, Jiasharete; Aibai, Minawa; Sabirhazi, Gulnur; Shawutali, Nuerai; Tangkejie, Wulanbai; Badelhan, Aynaz; Nuerduola, Yeermike; Satewalede, Turde; Buranbai, Darehan; Hunapia, Beicen; Jialihasi, Ayidaer; Bai, Jingping; Kizaibek, Murat

    2012-12-15

    Active Achilles tendon kinesitherapy facilitates the functional recovery of a ruptured Achilles tendon. However, protein expression during the healing process remains a controversial issue. New Zealand rabbits, aged 14 weeks, underwent tenotomy followed immediately by Achilles tendon microsurgery to repair the Achilles tendon rupture. The tendon was then immobilized or subjected to postoperative early motion treatment (kinesitherapy). Mass spectrography results showed that after 14 days of motion treatment, 18 protein spots were differentially expressed, among which, 12 were up-regulated, consisting of gelsolin isoform b and neurite growth-related protein collapsing response mediator protein 2. Western blot analysis showed that gelsolin isoform b was up-regulated at days 7-21 of motion treatment. These findings suggest that active Achilles tendon kinesitherapy promotes the neurite regeneration of a ruptured Achilles tendon and gelsolin isoform b can be used as a biomarker for Achilles tendon healing after kinesitherapy. PMID:25317130

  2. FGF inhibits neurite outgrowth over monolayers of astrocytes and fibroblasts expressing transfected cell adhesion molecules.

    PubMed

    Williams, E J; Mittal, B; Walsh, F S; Doherty, P

    1995-11-01

    We have cultured cerebellar neurons on monolayers of cortical astrocytes in control medium or medium containing recombinant basic fibroblast growth factor (FGF). FGF was found to inhibit neurite outgrowth, with a significant effect seen at 0.5 ng/ml and a maximal effect at 10 ng/ml. FGF increased the production of arachidonic acid (AA) in cerebellar neurons, and when added directly to cultures or generated endogenously via activation of phospholipase A2 using melittin, this second messenger could mimic the inhibitory effect of FGF. FGF and AA could also specifically inhibit neurite outgrowth stimulated by three cell adhesion molecules (NCAM, N-cadherin and L1) expressed in transfected fibroblasts, or in the case of L1 bound to a tissue culture substratum. These data demonstrate that, in certain cellular contexts, FGF can act as an inhibitory cue for axonal growth and that arachidonic acid is the second messenger responsible for this activity. We discuss the possibility that arachidonic acid inhibits neurite outgrowth by desensitising the second messenger pathway underlying neuronal responsiveness to cell adhesion molecules. PMID:8586663

  3. Cyclic AMP Stimulates Neurite Outgrowth of Lamprey Reticulospinal Neurons without Substantially Altering Their Biophysical Properties

    PubMed Central

    Pale, Timothée; Frisch, Emily B.; McClellan, Andrew D.

    2013-01-01

    Reticulospinal (RS) neurons are critical for initiation of locomotor behavior, and following spinal cord injury (SCI) in the lamprey, the axons of these neurons regenerate and restore locomotor behavior within a few weeks. For lamprey RS neurons in culture, experimental induction of calcium influx, either in the growth cone or cell body, is inhibitory for neurite outgrowth. Following SCI, these neurons partially downregulate calcium channel expression, which would be expected to reduce calcium influx and possibly provide supportive conditions for axonal regeneration. In the present study, it was tested whether activation of second messenger signaling pathways stimulates neurite outgrowth of lamprey RS neurons without altering their electrical properties (e.g. spike broadening) so as to possibly increase calcium influx and compromise axonal growth. First, activation of cAMP pathways with forskolin or dbcAMP stimulated neurite outgrowth of RS neurons in culture in a PKA-dependent manner, while activation of cGMP signaling pathways with dbcGMP inhibited outgrowth. Second, neurophysiological recordings from uninjured RS neurons in isolated lamprey brain-spinal cord preparations indicated that dbcAMP or dbcGMP did not significantly affect any of the measured electrical properties. In contrast, for uninjured RS neurons, forskolin increased action potential duration, which might have increased calcium influx, but did not significantly affect most other electrical properties. Importantly, for injured RS neurons during the period of axonal regeneration, forskolin did not significantly alter their electrical properties. Taken together, these results suggest that activation of cAMP signaling by dbcAMP stimulates neurite outgrowth, but does not alter the electrical properties of lamprey RS neurons in such a way that would be expected to induce calcium influx. In conclusion, our results suggest that activation of cAMP pathways alone, without compensation for possible

  4. Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth

    SciTech Connect

    Fukumitsu, Hidefumi; Soumiya, Hitomi; Furukawa, Shoei

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer CFIm25 knockdown promoted NGF-induced neurite out growth from PC12 cells. Black-Right-Pointing-Pointer Depletion of CFIm25 did not influence the morphology of proliferating PC12 cells. Black-Right-Pointing-Pointer CFIm regulated NGF-induced neurite outgrowth via coordinating RhoA activity. Black-Right-Pointing-Pointer CFIm25 knockdown increase the number of primary dendrites of hippocampal neurons. -- Abstract: Mammalian precursor mRNA (pre-mRNA) cleavage factor I (CFIm) plays important roles in the selection of poly(A) sites in a 3 Prime -untranslated region (3 Prime -UTR), producing mRNAs with variable 3 Prime ends. Because 3 Prime -UTRs often contain cis elements that impact stability or localization of mRNA or translation, alternative polyadenylation diversifies utilization of primary transcripts in mammalian cells. However, the physiological role of CFIm remains unclear. CFIm acts as a heterodimer comprising a 25 kDa subunit (CFIm25) and one of the three large subunits-CFIm59, CFIm68, or CFIm72. CFIm25 binds directly to RNA and introduces and anchors the larger subunit. To examine the physiological roles of CFIm, we knocked down the CFIm25 gene in neuronal cells using RNA interference. Knockdown of CFIm25 increased the number of primary dendrites of developing hippocampal neurons and promoted nerve growth factor (NGF)-induced neurite extension from rat pheochromocytoma PC12 cells without affecting the morphology of proliferating PC12 cells. On the other hand, CFIm25 knockdown did not influence constitutively active or dominantly negative RhoA suppression or promotion of NGF-induced neurite extension from PC12 cells, respectively. Taken together, our results indicate that endogenous CFIm may promote neuritogenesis in developing neurons by coordinating events upstream of NGF-induced RhoA inactivation.

  5. Experimental microembolism induces localized neuritic pathology in guinea pig cerebrum

    PubMed Central

    Li, Jian-Ming; Cai, Yan; Liu, Fei; Yang, La; Hu, Xia; Patrylo, Peter R.; Cai, Huaibin; Luo, Xue-Gang; Xiao, Dong; Yan, Xiao-Xin

    2015-01-01

    Microbleeds are a common finding in aged human brains. In Alzheimer's disease (AD), neuritic plaques composed of β-amyloid (Aβ) deposits and dystrophic neurites occur frequently around cerebral vasculature, raising a compelling question as to whether, and if so, how, microvascular abnormality and amyloid/neuritic pathology might be causally related. Here we used a guinea pig model of cerebral microembolism to explore a potential inductive effect of vascular injury on neuritic and amyloid pathogenesis. Brains were examined 7-30 days after experimental microvascular embolization occupying ~0.5% of total cortical area. Compared to sham-operated controls, glial fibrillary acidic protein immunoreactivity was increased in the embolized cerebrum, evidently around intracortical vasculature. Swollen/sprouting neurites exhibiting increased reactivity of nicotinamide adenine dinucleotide phosphate diaphorase, parvalbumin, vesicular glutamate transporter 1 and choline acetyltransferase appeared locally in the embolized brains in proximity to intracortical vasculature. The embolization-induced swollen/sprouting neurites were also robustly immunoreactive for β-amyloid precursor protein and β-secretase-1, the substrate and initiating enzyme for Aβ genesis. These experimental data suggest that microvascular injury can induce multisystem neuritic pathology associated with an enhanced amyloidogenic potential in wild-type mammalian brain. PMID:25871402

  6. Experimental microembolism induces localized neuritic pathology in guinea pig cerebrum.

    PubMed

    Li, Jian-Ming; Cai, Yan; Liu, Fei; Yang, La; Hu, Xia; Patrylo, Peter R; Cai, Huaibin; Luo, Xue-Gang; Xiao, Dong; Yan, Xiao-Xin

    2015-05-10

    Microbleeds are a common finding in aged human brains. In Alzheimer's disease (AD), neuritic plaques composed of β-amyloid (Aβ) deposits and dystrophic neurites occur frequently around cerebral vasculature, raising a compelling question as to whether, and if so, how, microvascular abnormality and amyloid/neuritic pathology might be causally related. Here we used a guinea pig model of cerebral microembolism to explore a potential inductive effect of vascular injury on neuritic and amyloid pathogenesis. Brains were examined 7-30 days after experimental microvascular embolization occupying ~0.5% of total cortical area. Compared to sham-operated controls, glial fibrillary acidic protein immunoreactivity was increased in the embolized cerebrum, evidently around intracortical vasculature. Swollen/sprouting neurites exhibiting increased reactivity of nicotinamide adenine dinucleotide phosphate diaphorase, parvalbumin, vesicular glutamate transporter 1 and choline acetyltransferase appeared locally in the embolized brains in proximity to intracortical vasculature. The embolization-induced swollen/sprouting neurites were also robustly immunoreactive for β-amyloid precursor protein and β-secretase-1, the substrate and initiating enzyme for Aβ genesis. These experimental data suggest that microvascular injury can induce multisystem neuritic pathology associated with an enhanced amyloidogenic potential in wild-type mammalian brain. PMID:25871402

  7. Mechanical stress activates neurites and somata of myenteric neurons

    PubMed Central

    Kugler, Eva M.; Michel, Klaus; Zeller, Florian; Demir, Ihsan E.; Ceyhan, Güralp O.; Schemann, Michael; Mazzuoli-Weber, Gemma

    2015-01-01

    The particular location of myenteric neurons, sandwiched between the 2 muscle layers of the gut, implies that their somata and neurites undergo mechanical stress during gastrointestinal motility. Existence of mechanosensitive enteric neurons (MEN) is undoubted but many of their basic features remain to be studied. In this study, we used ultra-fast neuroimaging to record activity of primary cultured myenteric neurons of guinea pig and human intestine after von Frey hair evoked deformation of neurites and somata. Independent component analysis was applied to reconstruct neuronal morphology and follow neuronal signals. Of the cultured neurons 45% (114 out of 256, 30 guinea pigs) responded to neurite probing with a burst spike frequency of 13.4 Hz. Action potentials generated at the stimulation site invaded the soma and other neurites. Mechanosensitive sites were expressed across large areas of neurites. Many mechanosensitive neurites appeared to have afferent and efferent functions as those that responded to deformation also conducted spikes coming from the soma. Mechanosensitive neurites were also activated by nicotine application. This supported the concept of multifunctional MEN. 14% of the neurons (13 out of 96, 18 guinea pigs) responded to soma deformation with burst spike discharge of 17.9 Hz. Firing of MEN adapted rapidly (RAMEN), slowly (SAMEN), or ultra-slowly (USAMEN). The majority of MEN showed SAMEN behavior although significantly more RAMEN occurred after neurite probing. Cultured myenteric neurons from human intestine had similar properties. Compared to MEN, dorsal root ganglion neurons were activated by neurite but not by soma deformation with slow adaptation of firing. We demonstrated that MEN exhibit specific features very likely reflecting adaptation to their specialized functions in the gut. PMID:26441520

  8. Mechanical stress activates neurites and somata of myenteric neurons.

    PubMed

    Kugler, Eva M; Michel, Klaus; Zeller, Florian; Demir, Ihsan E; Ceyhan, Güralp O; Schemann, Michael; Mazzuoli-Weber, Gemma

    2015-01-01

    The particular location of myenteric neurons, sandwiched between the 2 muscle layers of the gut, implies that their somata and neurites undergo mechanical stress during gastrointestinal motility. Existence of mechanosensitive enteric neurons (MEN) is undoubted but many of their basic features remain to be studied. In this study, we used ultra-fast neuroimaging to record activity of primary cultured myenteric neurons of guinea pig and human intestine after von Frey hair evoked deformation of neurites and somata. Independent component analysis was applied to reconstruct neuronal morphology and follow neuronal signals. Of the cultured neurons 45% (114 out of 256, 30 guinea pigs) responded to neurite probing with a burst spike frequency of 13.4 Hz. Action potentials generated at the stimulation site invaded the soma and other neurites. Mechanosensitive sites were expressed across large areas of neurites. Many mechanosensitive neurites appeared to have afferent and efferent functions as those that responded to deformation also conducted spikes coming from the soma. Mechanosensitive neurites were also activated by nicotine application. This supported the concept of multifunctional MEN. 14% of the neurons (13 out of 96, 18 guinea pigs) responded to soma deformation with burst spike discharge of 17.9 Hz. Firing of MEN adapted rapidly (RAMEN), slowly (SAMEN), or ultra-slowly (USAMEN). The majority of MEN showed SAMEN behavior although significantly more RAMEN occurred after neurite probing. Cultured myenteric neurons from human intestine had similar properties. Compared to MEN, dorsal root ganglion neurons were activated by neurite but not by soma deformation with slow adaptation of firing. We demonstrated that MEN exhibit specific features very likely reflecting adaptation to their specialized functions in the gut. PMID:26441520

  9. Nerve abscess in primary neuritic leprosy.

    PubMed

    Rai, Dheeraj; Malhotra, Hardeep Singh; Garg, Ravindra Kumar; Goel, Madhu Mati; Malhotra, Kiran Preet; Kumar, Vijay; Singh, Arun Kumar; Jain, Amita; Kohli, Neera; Singh, Shailesh Kumar

    2013-06-01

    Nerve abscess is an infrequently reported complication of leprosy. We describe a patient with a pure neuritic type of leprosy with multiple nerve abscesses, who presented with tingling and numbness in the medial aspect of his right forearm and hand. Subsequently he developed pain, redness and swelling over the medial side of his right elbow and the flexor aspect of his right wrist. High-resolution ultrasound showed diffuse thickening of the right ulnar nerve with hypoechoic texture housing a cystic lesion with internal debris suggesting an abscess, at the cubital tunnel. Histopathological examination of the pus and tissue obtained from the abscess revealed presence of granulomas with lepra bacilli. The patient responded to surgery and multidrug therapy. In conclusion, the nerve abscess as the first manifestation of leprosy is uncommon and a high index of suspicion is required to make a correct diagnosis. PMID:24171239

  10. NTAK/neuregulin-2 secreted by astrocytes promotes survival and neurite outgrowth of neurons via ErbB3.

    PubMed

    Nakano, Norihiko; Kanekiyo, Kenji; Nakagawa, Takatoshi; Asahi, Michio; Ide, Chizuka

    2016-05-27

    NTAK (neural- and thymus-derived activator for ErbB kinases), also known as neuregulin-2 (NRG2), is a member of the epidermal growth factor (EGF) family, which binds directly to ErbB3 and ErbB4, and transactivates ErbB2. NTAK/NRG2 is structurally homologous to NRG1. The biological function of NTAK/NRG2 still remains unknown, especially in the nervous system, whereas NRG1 is known to be essential for nervous system function. In the present study, we examined the functions of NTAK/NRG2 secreted from astrocytes to neurons. NTAK/NRG2 was expressed in both neurons and astrocytes, as evidenced by immunohistochemical staining and RT-PCR methods. The conditioned medium (CM) from astrocytes promoted survival and neurite outgrowth of neurons. The CM stimulated phosphorylation of ErbB3 in neurons. When phosphorylation of ErbB3 was blocked by AZD8931, an ErbB3 inhibitor, neuronal survival and neurite outgrowth were reduced. Conversely, canertinib, an ErbB4 inhibitor, did not affect survival or neurite outgrowth of neurons. Survival and neurite outgrowth of neurons were lower in CM of NTAK/NRG2-knockdown astrocytes than in the CM of control astrocytes, whereas the CM of NRG1-knockdown astrocytes had little effect on survival and neurite outgrowth. The present study demonstrated that NTAK/NRG2 secreted from astrocytes bound to ErbB3 on neurons, and promoted neuronal survival and neurite extension in vitro. PMID:27113200

  11. The functionalized amino acid (S)-Lacosamide subverts CRMP2-mediated tubulin polymerization to prevent constitutive and activity-dependent increase in neurite outgrowth

    PubMed Central

    Wilson, Sarah M.; Moutal, Aubin; Melemedjian, Ohannes K.; Wang, Yuying; Ju, Weina; François-Moutal, Liberty; Khanna, May; Khanna, Rajesh

    2014-01-01

    Activity-dependent neurite outgrowth is a highly complex, regulated process with important implications for neuronal circuit remodeling in development as well as in seizure-induced sprouting in epilepsy. Recent work has linked outgrowth to collapsin response mediator protein 2 (CRMP2), an intracellular phosphoprotein originally identified as axon guidance and growth cone collapse protein. The neurite outgrowth promoting function of CRMP2 is regulated by its phosphorylation state. In this study, depolarization (potassium chloride)-driven activity increased the level of active CRMP2 by decreasing its phosphorylation by GSK3β via a reduction in priming by Cdk5. To determine the contribution of CRMP2 in activity-driven neurite outgrowth, we screened a limited set of compounds for their ability to reduce neurite outgrowth but not modify voltage-gated sodium channel (VGSC) biophysical properties. This led to the identification of (S)-lacosamide ((S)-LCM), a stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Whereas (S)-LCM was ineffective in targeting VGSCs, the presumptive pharmacological targets of (R)-LCM, (S)-LCM was more efficient than (R)-LCM in subverting neurite outgrowth. Biomolecular interaction analyses revealed that (S)-LCM bound to wildtype CRMP2 with low micromolar affinity, similar to (R)-LCM. Through the use of this novel tool, the activity-dependent increase in neurite outgrowth observed following depolarization was characterized to be reliant on CRMP2 function. Knockdown of CRMP2 by siRNA in cortical neurons resulted in reduced CRMP2-dependent neurite outgrowth; incubation with (S)-LCM phenocopied this effect. Other CRMP2-mediated processes were unaffected. (S)-LCM subverted neurite outgrowth not by affecting the canonical CRMP2-tubulin association but rather by impairing the ability of CRMP2 to promote tubulin polymerization, events that are

  12. Neurite Mistargeting and Inverse Order of Intraretinal Vascular Plexus Formation Precede Subretinal Vascularization in Vldlr Mutant Mice

    PubMed Central

    Johnson, Verity; Xiang, Mengqing; Chen, Zhe; Junge, Harald J.

    2015-01-01

    In the retina blood vessels are required to support a high metabolic rate, however, uncontrolled vascular growth can lead to impaired vision and blindness. Subretinal vascularization (SRV), one type of pathological vessel growth, occurs in retinal angiomatous proliferation and proliferative macular telangiectasia. In these diseases SRV originates from blood vessels within the retina. We use mice with a targeted disruption in the Vldl-receptor (Vldlr) gene as a model to study SRV with retinal origin. We find that Vldlr mRNA is strongly expressed in the neuroretina, and we observe both vascular and neuronal phenotypes in Vldlr-/- mice. Unexpectedly, horizontal cell (HC) neurites are mistargeted prior to SRV in this model, and the majority of vascular lesions are associated with mistargeted neurites. In Foxn4-/- mice, which lack HCs and display reduced amacrine cell (AC) numbers, we find severe defects in intraretinal capillary development. However, SRV is not suppressed in Foxn4-/-;Vldlr-/- mice, which reveals that mistargeted HC neurites are not required for vascular lesion formation. In the absence of VLDLR, the intraretinal capillary plexuses form in an inverse order compared to normal development, and subsequent to this early defect, vascular proliferation is increased. We conclude that SRV in the Vldlr-/- model is associated with mistargeted neurites and that SRV is preceded by altered retinal vascular development. PMID:26177550

  13. Propolis Inhibits Neurite Outgrowth in Differentiating SH-SY5Y Human Neuroblastoma Cells.

    PubMed

    Kim, Han Bit; Yoo, Byung Sun

    2016-07-01

    Propolis is a multicomponent, active, complex resinous substance collected by honeybees from a variety of plant sources. We have studied the effect of propolis on neurite outgrowth of SH-SY5Y human neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). Propolis, at a concentration of 3 μg/mL, had no significant effect on the viability of differentiating SH-SY5Y cells. However, the neurite outgrowth of the differentiating SH-SY5Y cells treated with propolis (0.3~3 μg/mL) for 48 hr was significantly inhibited in a dose-dependent manner. Treatment of RA-stimulated differentiating SH-SY5Y cells with 0.3 to 3 μg/mL propolis resulted in decreased level of transglutaminase and 43-kDa growth-associated protein (GAP-43) in a dose-dependent manner. The results indicate that propolis is able to inhibit neurite outgrowth of differentiating SH-SY5Y cells. PMID:27437091

  14. Propolis Inhibits Neurite Outgrowth in Differentiating SH-SY5Y Human Neuroblastoma Cells

    PubMed Central

    Kim, Han Bit; Yoo, Byung Sun

    2016-01-01

    Propolis is a multicomponent, active, complex resinous substance collected by honeybees from a variety of plant sources. We have studied the effect of propolis on neurite outgrowth of SH-SY5Y human neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). Propolis, at a concentration of 3 μg/mL, had no significant effect on the viability of differentiating SH-SY5Y cells. However, the neurite outgrowth of the differentiating SH-SY5Y cells treated with propolis (0.3~3 μg/mL) for 48 hr was significantly inhibited in a dose-dependent manner. Treatment of RA-stimulated differentiating SH-SY5Y cells with 0.3 to 3 μg/mL propolis resulted in decreased level of transglutaminase and 43-kDa growth-associated protein (GAP-43) in a dose-dependent manner. The results indicate that propolis is able to inhibit neurite outgrowth of differentiating SH-SY5Y cells. PMID:27437091

  15. Effects of borate-based bioactive glass on neuron viability and neurite extension.

    PubMed

    Marquardt, Laura M; Day, Delbert; Sakiyama-Elbert, Shelly E; Harkins, Amy B

    2014-08-01

    Bioactive glasses have recently been shown to promote regeneration of soft tissues by positively influencing tissue remodeling during wound healing. We were interested to determine whether bioactive glasses have the potential for use in the treatment of peripheral nerve injury. In these experiments, degradable bioactive borate glass was fabricated into rods and microfibers. To study the compatibility with neurons, embryonic chick dorsal root ganglia (DRG) were cultured with different forms of bioactive borate glass. Cell viability was measured with no media exchange (static condition) or routine media exchange (transient condition). Neurite extension was measured within fibrin scaffolds with embedded glass microfibers or aligned rod sheets. Mixed cultures of neurons, glia, and fibroblasts growing in static conditions with glass rods and microfibers resulted in decreased cell viability. However, the percentage of neurons compared with all cell types increased by the end of the culture protocol compared with culture without glass. Furthermore, bioactive glass and fibrin composite scaffolds promoted neurite extension similar to that of control fibrin scaffolds, suggesting that glass does not have a significant detrimental effect on neuronal health. Aligned glass scaffolds guided neurite extension in an oriented manner. Together these findings suggest that bioactive glass can provide alignment to support directed axon growth. PMID:24027222

  16. Neurite formation by neurons derived from adult rat hippocampal progenitor cells is susceptible to myelin inhibition.

    PubMed

    Mellough, Carla B; Cho, Seongeun; Wood, Andrew; Przyborski, Stefan

    2011-09-01

    Myelin-associated inhibitors expressed following injury to the adult central nervous system (CNS) induce growth cone collapse and retraction of the axonal cytoskeleton. Myelin-associated glycoprotein (MAG) is a bi-functional molecule that promotes neuritogenesis in some immature neurons during development then becomes inhibitory to neurite outgrowth as neurons mature. Progress is being made towards the elucidation of the downstream events that regulate myelin inhibition of regeneration in neuronal populations. However it is not known how adult-derived neural stem cells or progenitors respond to myelin during neuronal differentiation and neuritogenesis. Here we examine the effect of MAG on neurons derived from an adult rat hippocampal progenitor cell line (AHPCs). We show that, unlike their developmental counterparts, AHPC-derived neurons are susceptible to MAG inhibition of neuritogenesis during differentiation and display a 57% reduction in neurite outgrowth when compared with controls. We demonstrate that this effect can be overcome (by up to 69%) by activation of the neurotrophin, cyclic AMP and protein kinase A pathways or by Rho-kinase suppression. We also demonstrate that combination of these factors enhanced neurite outgrowth from differentiating neurons in the presence of MAG. This work provides important information for the successful generation of new neurons from adult neural stem cell populations within compromised adult circuitry and is thus directly relevant to endogenous repair and regeneration of the adult CNS. PMID:21256909

  17. Influence of micro-patterned PLLA membranes on outgrowth and orientation of hippocampal neurites.

    PubMed

    Morelli, Sabrina; Salerno, Simona; Piscioneri, Antonella; Papenburg, Bernke J; Di Vito, Anna; Giusi, Giuseppina; Canonaco, Marcello; Stamatialis, Dimitrios; Drioli, Enrico; De Bartolo, Loredana

    2010-09-01

    In neuronal tissue engineering many efforts are focused on creating biomaterials with physical and chemical pathways for controlling cellular proliferation and orientation. Neurons have the ability to respond to topographical features in their microenvironment causing among others, axons to proliferate along surface features such as substrate grooves in micro-and nanoscales. As a consequence these neuronal elements are able to correctly adhere, migrate and orient within their new environment during growth. Here we explored the polarization and orientation of hippocampal neuronal cells on nonpatterned and micro-patterned biodegradable poly(l-lactic acid) (PLLA) membranes with highly selective permeable properties. Dense and porous nonpatterned and micro-patterned membranes were prepared from PLLA by Phase Separation Micromolding. The micro-patterned membranes have a three-dimensional structure consisting of channels and ridges and of bricks of different widths. Nonpatterned and patterned membranes were used for hippocampal neuronal cultures isolated from postnatal days 1-3 hamsters and the neurite length, orientation and specific functions of cells were investigated up to 12 days of culture. Neurite outgrowth, length plus orientation tightly overlapped the pattern of the membrane surface. Cell distribution occurred only in correspondence to membrane grooves characterized by continuous channels whereas on membranes with interconnected channels, cells not only adhered to and elongated their cellular processes in the grooves but also in the breaking points. High orientation degrees of cells were determined particularly on the patterned porous membranes with channel width of 20 mum and ridges of 17 mum whereas on dense nonpatterned membranes as well as on polystyrene culture dish (PSCD) controls, a larger number of primary developed neurites were distributed. Based on these results, PLLA patterned membranes may directly improve the guidance of neurite extension and

  18. Pulsed electromagnetic fields potentiate neurite outgrowth in the dopaminergic MN9D cell line.

    PubMed

    Lekhraj, Rukmani; Cynamon, Deborah E; DeLuca, Stephanie E; Taub, Eric S; Pilla, Arthur A; Casper, Diana

    2014-06-01

    Pulsed electromagnetic fields (PEMF) exert biological effects and are in clinical use to facilitate bone repair and wound healing. Research has demonstrated that PEMF can induce signaling molecules and growth factors, molecules that play important roles in neuronal differentiation. Here, we tested the effects of a low-amplitude, nonthermal, pulsed radiofrequency signal on morphological neuronal differentiation in MN9D, a dopaminergic cell line. Cells were plated in medium with 10% fetal calf serum. After 1 day, medium was replaced with serum-containing medium, serum-free medium, or medium supplemented with dibutyryl cyclic adenosine monophosphate (Bt2 cAMP), a cAMP analog known to induce neurite outgrowth. Cultures were divided into groups and treated with PEMF signals for either 30 min per day or continuously for 15 min every hour for 3 days. Both serum withdrawal and Bt2 cAMP significantly increased neurite length. PEMF treatment similarly increased neurite length under both serum-free and serum-supplemented conditions, although to a lesser degree in the presence of serum, when continuous treatments had greater effects. PEMF signals also increased cell body width, indicating neuronal maturation, and decreased protein content, suggesting that this treatment was antimitotic, an effect reversed by the inhibitor of cAMP formation dideoxyadenosine. Bt2 cAMP and PEMF effects were not additive, suggesting that neurite elongation was achieved through a common pathway. PEMF signals increased cAMP levels from 3 to 5 hr after treatment, supporting this mechanism of action. Although neuritogenesis is considered a developmental process, it may also represent the plasticity required to form and maintain synaptic connections throughout life. PMID:24523147

  19. Analysis of slow-onset neurite formation in NG108-15 cells: implications for a unified model of neurite elongation.

    PubMed

    Smalheiser, N R

    1989-01-01

    When undifferentiated NG108-15 cells are plated onto polylysine coated Petri dishes in serum-free medium, they form neurites within 1-4 h if plated in the presence of laminin or 5'-deoxy-5'-methylthioadenosine (rapid-onset neurites). In the absence of such agents, serum-deprived NG108-15 cells extend axon-like neurites onto polylysine over several days; here we characterize the dynamic behavior of this slow-onset outgrowth pattern in detail. Individual cells plated on laminin expressed a gradual multipolar-to-unipolar transition due to rapid-onset neurites becoming remodelled into the appearance of slow-onset neurites. This phenomenon reflected the selective stabilization of certain rapid-onset neurites, along with the restriction of motility to their distal tips. Based upon the properties and interactions of both rapid- and slow-onset neurites in NG108-15 cells, a unified model for neurite formation is presented. PMID:2917412

  20. 7, 8, 3′-Trihydroxyflavone Promotes Neurite Outgrowth and Protects Against Bupivacaine-Induced Neurotoxicity in Mouse Dorsal Root Ganglion Neurons

    PubMed Central

    Shi, Haohong; Luo, Xingjing

    2016-01-01

    Background 7, 8, 3′-trihydroxyflavone (THF) is a novel pro-neuronal small molecule that acts as a TrkB agonist. In this study, we examined the effect of THF on promoting neuronal growth and protecting anesthetics-induced neurotoxicity in dorsal root ganglion (DRG) neurons in vitro. Material/Methods Neonatal mouse DRG neurons were cultured in vitro and treated with various concentrations of THF. The effect of THF on neuronal growth was investigated by neurite outgrowth assay and Western blot. In addition, the protective effects of THF on bupivacaine-induced neurotoxicity were investigated by apoptosis TUNEL assay, neurite outgrowth assay, and Western blot, respectively. Results THF promoted neurite outgrowth of DRG neurons in dose-dependent manner, with an EC50 concentration of 67.4 nM. Western blot analysis showed THF activated TrkB signaling pathway by inducing TrkB phosphorylation. THF also rescued bupivacaine-induced neurotoxicity by reducing apoptosis and protecting neurite retraction in DRG neurons. Furthermore, the protection of THF in bupivacaine-injured neurotoxicity was directly associated with TrkB phosphorylation in a concentration-dependent manner in DRG neurons. Conclusions THF has pro-neuronal effect on DRG neurons by promoting neurite growth and protecting against bupivacaine-induced neurotoxicity, likely through TrkB activation. PMID:27371503

  1. Angiopoietin-1 induces neurite outgrowth of PC12 cells in a Tie2-independent, β1-integrin dependent manner

    PubMed Central

    Chen, Xinyu; Fu, Wen; Tung, Christie E.; Ward, Nicole L.

    2009-01-01

    Overexpression of Angiopoietin (Ang) 1 in the brain results in increased vascularization and altered neuronal dendrite configuration. We hypothesized that Ang1 acts directly on neurons inducing neurite outgrowth. We stimulated PC12 cells with Ang1 and observed outgrowth levels comparable to nerve growth factor (NGF). Western blotting and RT-PCR demonstrated the absence of the Ang1 receptor, Tie2 and the presence of β1-integrin. Downstream of β1-integrin, Ang1 stimulation led to a ~2.6 fold increase in focal adhesion kinase (FAK) phosphorylation and no change in activation of mitogen-activated protein kinase (MAPK) nor c-Jun N-terminal kinase (JNK). Conversely, NGF stimulation had no effect on FAK phosphorylation but lead to a ~3.1 and ~2 fold increase in phosphorylation of MAPK and JNK. Ang1, but not NGF-mediated outgrowth was attenuated following functional inhibition of β1-integrin and FAK, and Wortmannin inhibited neurite outgrowth mediated by both. Our results suggest that Ang1 induces neurite outgrowth in PC12 cells in a Tie2-independent, β1-integrin-FAK-PI3K-Akt dependent manner and that NGF and Ang1 mediate neurite outgrowth via two independent signaling mechanisms. PMID:19379779

  2. White Matter Changes of Neurite Density and Fiber Orientation Dispersion during Human Brain Maturation

    PubMed Central

    Chang, Yi Shin; Owen, Julia P.; Pojman, Nicholas J.; Thieu, Tony; Bukshpun, Polina; Wakahiro, Mari L. J.; Berman, Jeffrey I.; Roberts, Timothy P. L.; Nagarajan, Srikantan S.; Sherr, Elliott H.; Mukherjee, Pratik

    2015-01-01

    Diffusion tensor imaging (DTI) studies of human brain development have consistently shown widespread, but nonlinear increases in white matter anisotropy through childhood, adolescence, and into adulthood. However, despite its sensitivity to changes in tissue microstructure, DTI lacks the specificity to disentangle distinct microstructural features of white and gray matter. Neurite orientation dispersion and density imaging (NODDI) is a recently proposed multi-compartment biophysical model of brain microstructure that can estimate non-collinear properties of white matter, such as neurite orientation dispersion index (ODI) and neurite density index (NDI). In this study, we apply NODDI to 66 healthy controls aged 7–63 years to investigate changes of ODI and NDI with brain maturation, with comparison to standard DTI metrics. Using both region-of-interest and voxel-wise analyses, we find that NDI exhibits striking increases over the studied age range following a logarithmic growth pattern, while ODI rises following an exponential growth pattern. This novel finding is consistent with well-established age-related changes of FA over the lifespan that show growth during childhood and adolescence, plateau during early adulthood, and accelerating decay after the fourth decade of life. Our results suggest that the rise of FA during the first two decades of life is dominated by increasing NDI, while the fall in FA after the fourth decade is driven by the exponential rise of ODI that overcomes the slower increases of NDI. Using partial least squares regression, we further demonstrate that NODDI better predicts chronological age than DTI. Finally, we show excellent test—retest reliability of NODDI metrics, with coefficients of variation below 5% in all measured regions of interest. Our results support the conclusion that NODDI reveals biologically specific characteristics of brain development that are more closely linked to the microstructural features of white matter than

  3. Vascular endothelial growth factor induces anti‑Müllerian hormone receptor 2 overexpression in ovarian granulosa cells of in vitro fertilization/intracytoplasmic sperm injection patients.

    PubMed

    Fang, Yanqiu; Lu, Xiaodan; Liu, Lei; Lin, Xiuying; Sun, Munan; Fu, Jianhua; Xu, Shufen; Tan, Yan

    2016-06-01

    Misregulation of vascular endothelial growth factor A (VEGF‑A) has been implicated in numerous types of ovarian disease, such as polycystic ovarian syndrome, ovarian hyperstimulation syndrome, endometriosis and ovarian cancer. VEGF regulates blood vessel permeability and angiogenesis. In our previous study, VEGF‑regulated gene expression was profiled in the uterus of a transgenic mouse model with repressed VEGF expression, which indicated that VEGF is an important regulator in controlling gene expression in the uterus. The anti‑Müllerian hormone (AMH) is expressed by ovarian granulosa cells (GCs) and acts through its type 2 receptor, AMH receptor 2 (AMHR2). Serum AMH levels are used to predict ovarian reserves and the small antral follicles contribute markedly to the serum AMH level. AMH recruits primordial follicles and inhibits excessive follicular development by follicular stimulating hormone (FSH). However, AMH may be influenced by suppression of gonadotrophin secretion and VEGF inhibition. In the current study, human primary ovarian GCs were isolated from ovarian follicle fluid of in vitro fertilization/intracytoplasmic sperm injection cycles (IVF/ICSI). It was identified that the FSH receptor was consistently expressed in the isolated cells. VEGF‑A treatment stimulated AMHR2 overexpression at the gene and protein levels. In addition, VEGF induced AMHR2 expression on the surface of the isolated GCs from mature follicles. The VEGF treatment was also performed in an ovarian granulosa‑like cell line, KGN. AMH and AMHR2 are co‑expressed in normal GCs; however, as a result of VEGF misregulation, AMHR2 overexpression increases AMH binding, which may attenuate follicular or oocyte maturation. However, the associated function and underlying mechanism requires further investigation. PMID:27109000

  4. β2-Glycoprotein I Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Suppressing the Phosphorylation of Extracellular Signal-Regulated Kinase 1/2, Akt, and Endothelial Nitric Oxide Synthase.

    PubMed

    Chiu, Wen-Chin; Chiou, Tzeon-Jye; Chung, Meng-Ju; Chiang, An-Na

    2016-01-01

    Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The β2-glycoprotein I (β2-GPI) is a plasma glycoprotein with multiple biological functions, some of which remain to be elucidated. This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that β2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both β2-GPI and deglycosylated β2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of β2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that β2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, β2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that β2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for β2-GPI in anti-angiogenic therapy. PMID:27579889

  5. 7,3',4'-Trihydroxyisoflavone inhibits epidermal growth factor-induced proliferation and transformation of JB6 P+ mouse epidermal cells by suppressing cyclin-dependent kinases and phosphatidylinositol 3-kinase.

    PubMed

    Lee, Dong Eun; Lee, Ki Won; Song, Nu Ry; Seo, Sang Kwon; Heo, Yong-Seok; Kang, Nam Joo; Bode, Ann M; Lee, Hyong Joo; Dong, Zigang

    2010-07-01

    Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents. PMID:20444693

  6. Neurite, a Finite Difference Large Scale Parallel Program for the Simulation of Electrical Signal Propagation in Neurites under Mechanical Loading

    PubMed Central

    García-Grajales, Julián A.; Rucabado, Gabriel; García-Dopico, Antonio; Peña, José-María; Jérusalem, Antoine

    2015-01-01

    With the growing body of research on traumatic brain injury and spinal cord injury, computational neuroscience has recently focused its modeling efforts on neuronal functional deficits following mechanical loading. However, in most of these efforts, cell damage is generally only characterized by purely mechanistic criteria, functions of quantities such as stress, strain or their corresponding rates. The modeling of functional deficits in neurites as a consequence of macroscopic mechanical insults has been rarely explored. In particular, a quantitative mechanically based model of electrophysiological impairment in neuronal cells, Neurite, has only very recently been proposed. In this paper, we present the implementation details of this model: a finite difference parallel program for simulating electrical signal propagation along neurites under mechanical loading. Following the application of a macroscopic strain at a given strain rate produced by a mechanical insult, Neurite is able to simulate the resulting neuronal electrical signal propagation, and thus the corresponding functional deficits. The simulation of the coupled mechanical and electrophysiological behaviors requires computational expensive calculations that increase in complexity as the network of the simulated cells grows. The solvers implemented in Neurite—explicit and implicit—were therefore parallelized using graphics processing units in order to reduce the burden of the simulation costs of large scale scenarios. Cable Theory and Hodgkin-Huxley models were implemented to account for the electrophysiological passive and active regions of a neurite, respectively, whereas a coupled mechanical model accounting for the neurite mechanical behavior within its surrounding medium was adopted as a link between electrophysiology and mechanics. This paper provides the details of the parallel implementation of Neurite, along with three different application examples: a long myelinated axon, a segmented

  7. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    SciTech Connect

    Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R.

    2011-11-15

    There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

  8. Pleurotus giganteus (Berk.) Karunarathna & K.D. Hyde: Nutritional value and in vitro neurite outgrowth activity in rat pheochromocytoma cells

    PubMed Central

    2012-01-01

    Background Drugs dedicated to alleviate neurodegenerative diseases like Parkinson’s and Alzheimer’s have always been associated with debilitating side effects. Medicinal mushrooms which harness neuropharmacological compounds offer a potential possibility for protection against such diseases. Pleurotus giganteus (formerly known as Panus giganteus) has been consumed by the indigenous people in Peninsular Malaysia for many years. Domestication of this wild mushroom is gaining popularity but to our knowledge, medicinal properties reported for this culinary mushroom are minimal. Methods The fruiting bodies P. giganteus were analysed for its nutritional values. Cytotoxicity of the mushroom’s aqueous and ethanolic extracts towards PC12, a rat pheochromocytoma cell line was assessed by using 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Neurite outgrowth stimulation assay was carried out with nerve growth factor (NGF) as control. To elucidate signaling mechanisms involved by mushroom extract-induced neurite outgrowth, treatment of specific inhibitor for MEK/ERK and PI3K signalling pathway was carried out. Results The fruiting bodies of P. giganteus were found to have high carbohydrate, dietary fibre, potassium, phenolic compounds and triterpenoids. Both aqueous and ethanolic extracts induced neurite outgrowth of PC12 cells in a dose- and time-dependant manner with no detectable cytotoxic effect. At day 3, 25 μg/ml of aqueous extract and 15 μg/ml of ethanolic extract showed the highest percentage of neurite-bearing cells, i.e. 31.7 ± 1.1% and 33.3 ± 0.9%; respectively. Inhibition treatment results suggested that MEK/ERK and PI3K/Akt are responsible for neurite outgrowth of PC12 cells stimulated by P. giganteus extract. The high potassium content (1345.7 mg/100 g) may be responsible for promoting neurite extension, too. Conclusions P. giganteus contains bioactive compounds that mimic NGF and are responsible for neurite

  9. Rapid neurite outgrowth in neurosecretory cells and neurons is sustained by the exocytosis of a cytoplasmic organelle, the enlargeosome.

    PubMed

    Racchetti, Gabriella; Lorusso, Anna; Schulte, Carsten; Gavello, Daniela; Carabelli, Valentina; D'Alessandro, Rosalba; Meldolesi, Jacopo

    2010-01-15

    Neurite outgrowth is known as a slow (days) process occurring in nerve cells and neurons during neurotrophin treatment and upon transfer to culture, respectively. Using Y27632, a drug that induces activation of Rac1, a downstream step of the neurotrophin signaling cascade, we have identified a new form of outgrowth, which is rapid (<1 hour) and extensive (>500 microm(2) surface enlargement/single cell/first hour). However, this outgrowth takes place only in cells (PC12-27 and SH-SY5Y cells, and embryonic and neonatal neurons) rich in an exocytic organelle, the enlargeosome. Golgi vesicles, TGN vesicles and endosomes are not involved. The need for enlargeosomes for plasma-membrane expansion was confirmed by the appearance of their marker, Ahnak, at the cell surface and by the dependence of neurite outgrowth on VAMP4, the vSNARE of enlargeosome exocytosis. In enlargeosome-rich cells, VAMP4 downregulation also attenuated the slow outgrowth induced by nerve growth factor (NGF). Similar to NGF-induced neurite outgrowth in enlargeosome-lacking cells, the new, rapid, Y27632-induced process required microtubules. Other properties of neurite outgrowth in cells lacking enlargeosomes - such as dependence on VAMP7, on microfilaments, on gene transcription and on protein synthesis, and blockade of mitoses and accumulation of neuronal markers - were not evident. The enlargeosome-sustained process might be useful for the rapid neurite outgrowth at peculiar stages and/or conditions of nerve and neuronal cells. However, its properties and its physiological and pathological role remain to be investigated. PMID:20026640

  10. Interaction of new antidepressants with sigma-1 receptor chaperones and their potentiation of neurite outgrowth in PC12 cells.

    PubMed

    Ishima, Tamaki; Fujita, Yuko; Hashimoto, Kenji

    2014-03-15

    The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [(3)H](+)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine>sertraline>fluoxetine>escitalopram>citalopram>paroxetine>duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5-hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect. PMID:24508523

  11. Painful, degenerating intervertebral discs up-regulate neurite sprouting and CGRP through nociceptive factors.

    PubMed

    Krock, Emerson; Rosenzweig, Derek H; Chabot-Doré, Anne-Julie; Jarzem, Peter; Weber, Michael H; Ouellet, Jean A; Stone, Laura S; Haglund, Lisbet

    2014-06-01

    Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme-linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor-α, interleukin-1β, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene-related peptide expression, both of which were blocked by anti-NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo. PMID:24650225

  12. Painful, degenerating intervertebral discs up-regulate neurite sprouting and CGRP through nociceptive factors

    PubMed Central

    Krock, Emerson; Rosenzweig, Derek H; Chabot-Doré, Anne-Julie; Jarzem, Peter; Weber, Michael H; Ouellet, Jean A; Stone, Laura S; Haglund, Lisbet

    2014-01-01

    Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme-linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor-α, interleukin-1β, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene-related peptide expression, both of which were blocked by anti-NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo. PMID:24650225

  13. MiR-93 Targeting EphA4 Promotes Neurite Outgrowth from Spinal Cord Neurons.

    PubMed

    Chen, Xiaogang; Yang, Huilin; Zhou, Xiaoqing; Zhang, Lin; Lu, Xiaoqing

    2016-04-01

    The failure of neurite outgrowth in the adult mammalian spinal cord injury is thought to be attributed to the intrinsic growth ability of mature neurons. Ephrin/Eph system is a major growth regulator of many axonal guidance processes. EphA4 is expressed specifically in traumatic central nervous system (CNS) and dynamically regulate target gene expression, suggesting that it may be associated with neural regeneration. Here, we found an alteration in temporal expression of miR-93 following a contusive spinal cord injury (SCI) in adult rats. The messenger RNA (mRNA) expression level of miR-93 was upregulated and the protein expression levels of EphA4, p-Ephexin, and active RhoA were all decreased in traumatic spinal cord relative to those with an intact spinal cord. Infection of cultured spinal cord neurons (SCNs) with miR-93 mimic led to neuronal growth promotion and decreased levels of EphA4, p-Ephexin, and active RhoA protein expression. Dual-luciferase reporter assay confirmed that miR-93 bound to the three prime untranslated region (3' UTR) of EphA4 and inhibited the expression of EphA4 mRNA. These findings provide evidence that miR-93 inhibits EphA4 expression, decreased EphA4 expression could promote neurite outgrowth in SCNs due to reduced levels of p-Ephexin and active RhoA. PMID:26798048

  14. N -Glycans on the receptor for advanced glycation end products influence amphoterin binding and neurite outgrowth.

    PubMed

    Srikrishna, Geetha; Huttunen, Henri J; Johansson, Lena; Weigle, Bernd; Yamaguchi, Yu; Rauvala, Heikki; Freeze, Hudson H

    2002-03-01

    In this study we show that embryonic neurite growth-promoting protein amphoterin binds to carboxylated N -glycans previously identified on mammalian endothelial cells. Since amphoterin is a ligand for the receptor for advanced glycation end products (RAGE), and the ligand-binding V-domain of the receptor contains two potential N -glycosylation sites, we hypothesized that N -glycans on RAGE may mediate its interactions with amphoterin. In support of this, anti-carboxylate antibody mAbGB3.1 immunoprecipitates bovine RAGE, and PNGase F treatment reduces its molecular mass by 4.5 kDa, suggesting that the native receptor is a glycoprotein. The binding potential of amphoterin to RAGE decreases significantly in presence of soluble carboxylated glycans or when the receptor is deglycosylated. Oligosaccharide analysis shows that RAGE contains complex type anionic N -glycans with non-sialic acid carboxylate groups, but not the HNK-1 (3-sulfoglucuronyl beta1-3 galactoside) epitope. Consistent with the functional localization of RAGE and amphoterin at the leading edges of developing neurons, mAbGB3.1 stains axons and growth cones of mouse embryonic cortical neurons, and inhibits neurite outgrowth on amphoterin matrix. The carboxylated glycans themselves promote neurite outgrowth in embryonic neurons and RAGE-transfected neuroblastoma cells. This outgrowth requires full-length, signalling-competent RAGE, as cells expressing cytoplasmic domain-deleted RAGE are unresponsive. These results indicate that carboxylated N -glycans on RAGE play an important functional role in amphoterin-RAGE-mediated signalling. PMID:11953450

  15. Shoc2/Sur8 Protein Regulates Neurite Outgrowth

    PubMed Central

    Leon, Gonzalo; Sanchez-Ruiloba, Lucia; Perez-Rodriguez, Andrea; Gragera, Teresa; Martinez, Natalia; Hernandez, Silvia; Anta, Berta; Calero, Olga; Garcia-Dominguez, Carlota A.; Dura, Lara M.; Peña-Jimenez, Daniel; Castro, Judit; Zarich, Natasha; Sanchez-Gomez, Pilar; Calero, Miguel; Iglesias, Teresa; Oliva, Jose L.; Rojas, Jose M.

    2014-01-01

    The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth. PMID:25514808

  16. Neuroprotective copper bis(thiosemicarbazonato) complexes promote neurite elongation.

    PubMed

    Bica, Laura; Liddell, Jeffrey R; Donnelly, Paul S; Duncan, Clare; Caragounis, Aphrodite; Volitakis, Irene; Paterson, Brett M; Cappai, Roberto; Grubman, Alexandra; Camakaris, James; Crouch, Peter J; White, Anthony R

    2014-01-01

    Abnormal biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and motor neuron disease. Recent studies have shown that metal complexing compounds behaving as ionophores such as clioquinol and PBT2 have robust therapeutic activity in animal models of neurodegenerative disease; however, the mechanism of neuroprotective action remains unclear. These neuroprotective or neurogenerative processes may be related to the delivery or redistribution of biometals, such as copper and zinc, by metal ionophores. To investigate this further, we examined the effect of the bis(thiosemicarbazonato)-copper complex, Cu(II)(gtsm) on neuritogenesis and neurite elongation (neurogenerative outcomes) in PC12 neuronal-related cultures. We found that Cu(II)(gtsm) induced robust neurite elongation in PC12 cells when delivered at concentrations of 25 or 50 nM overnight. Analogous effects were observed with an alternative copper bis(thiosemicarbazonato) complex, Cu(II)(atsm), but at a higher concentration. Induction of neurite elongation by Cu(II)(gtsm) was restricted to neurites within the length range of 75-99 µm with a 2.3-fold increase in numbers of neurites in this length range with 50 nM Cu(II)(gtsm) treatment. The mechanism of neurogenerative action was investigated and revealed that Cu(II)(gtsm) inhibited cellular phosphatase activity. Treatment of cultures with 5 nM FK506 (calcineurin phosphatase inhibitor) resulted in analogous elongation of neurites compared to 50 nM Cu(II)(gtsm), suggesting a potential link between Cu(II)(gtsm)-mediated phosphatase inhibition and neurogenerative outcomes. PMID:24587210

  17. The role of calsyntenin-3 in dystrophic neurite formation in Alzheimer's disease brain.

    PubMed

    Uchida, Yoko; Gomi, Fujiya

    2016-03-01

    β-Amyloid (Aβ) oligomers may play an important role in the early pathogenesis of Alzheimer's disease: cognitive impairment caused by synaptic dysfunction. Dystrophic neurites surrounding Aβ plaques, another pathological feature of Alzheimer's disease, are plaque-associated neuritic alterations preceding the appearance of synaptic loss. In the present review, we focus on the mechanism of dystrophic neurite formation by Aß oligomers, and discuss the neurotoxic role of Aβ-induced calsyntenin-3 in mediating dystrophic neurite formation. PMID:27018282

  18. Micropatterned coumarin polyester thin films direct neurite orientation.

    PubMed

    McCormick, Aleesha M; Maddipatla, Murthy V S N; Shi, Shuojia; Chamsaz, Elaheh A; Yokoyama, Hiroshi; Joy, Abraham; Leipzig, Nic D

    2014-11-26

    Guidance and migration of cells in the nervous system is imperative for proper development, maturation, and regeneration. In the peripheral nervous system (PNS), it is challenging for axons to bridge critical-sized injury defects to achieve repair and the central nervous system (CNS) has a very limited ability to regenerate after injury because of its innate injury response. The photoreactivity of the coumarin polyester used in this study enables efficient micropatterning using a custom digital micromirror device (DMD) and has been previously shown to be biodegradable, making these thin films ideal for cell guidance substrates with potential for future in vivo applications. With DMD, we fabricated coumarin polyester thin films into 10×20 μm and 15×50 μm micropatterns with depths ranging from 15 to 20 nm to enhance nervous system cell alignment. Adult primary neurons, oligodendrocytes, and astrocytes were isolated from rat brain tissue and seeded onto the polymer surfaces. After 24 h, cell type and neurite alignment were analyzed using phase contrast and fluorescence imaging. There was a significant difference (p<0.0001) in cell process distribution for both emergence angle (from the body of the cell) and orientation angle (at the tip of the growth cone) confirming alignment on patterned surfaces compared to control substrates (unpatterned polymer and glass surfaces). The expected frequency distribution for parallel alignment (≤15°) is 14% and the two micropatterned groups ranged from 42 to 49% alignment for emergence and orientation angle measurements, where the control groups range from 12 to 22% for parallel alignment. Despite depths being 15 to 20 nm, cell processes could sense these topographical changes and preferred to align to certain features of the micropatterns like the plateau/channel interface. As a result this initial study in utilizing these new DMD micropatterned coumarin polyester thin films has proven beneficial as an axon guidance platform

  19. Berberine, a natural antidiabetes drug, attenuates glucose neurotoxicity and promotes Nrf2-related neurite outgrowth

    SciTech Connect

    Hsu, Ya-Yun; Tseng, Yu-Ting; Lo, Yi-Ching

    2013-11-01

    Reactive oxygen intermediates production and apoptotic damage induced by high glucose are major causes of neuronal damage in diabetic neuropathy. Berberine (BBR), a natural antidiabetes drug with PI3K-activating activity, holds promise for diabetes because of its dual antioxidant and anti-apoptotic activities. We have previously reported that BBR attenuated H{sub 2}O{sub 2} neurotoxicity via activating the PI3K/Akt/Nrf2-dependent pathway. In this study, we further explored the novel protective mechanism of BBR on high glucose-induced apoptotic death and neurite damage of SH-SY5Y cells. Results indicated BBR (0.1–10 nM) significantly attenuated reactive oxygen species (ROS) production, nucleus condensation, and apoptotic death in high glucose-treated cells. However, AG1024, an inhibitor of insulin growth factor-1 (IGF-1) receptor, significantly abolished BBR protection against high glucose-induced neuronal death. BBR also increased Bcl-2 expression and decreased cytochrome c release. High glucose down-regulated IGF-1 receptor and phosphorylation of Akt and GSK-3β, the effects of which were attenuated by BBR treatment. BBR also activated nuclear erythroid 2-related factor 2 (Nrf2), the key antioxidative transcription factor, which is accompanied with up-regulation of hemeoxygenase-1 (HO-1). Furthermore, BBR markedly enhanced nerve growth factor (NGF) expression and promoted neurite outgrowth in high glucose-treated cells. To further determine the role of the Nrf2 in BBR neuroprotection, RNA interference directed against Nrf2 was used. Results indicated Nrf2 siRNA abolished BBR-induced HO-1, NGF, neurite outgrowth and ROS decrease. In conclusion, BBR attenuated high glucose-induced neurotoxicity, and we are the first to reveal this novel mechanism of BBR as an Nrf2 activator against glucose neurotoxicity, providing another potential therapeutic use of BBR on the treatment of diabetic complications. - Highlights: • BBR attenuates high glucose-induced ROS

  20. Nur77 Was Essential for Neurite Outgrowth and Involved in Schwann Cell Differentiation After Sciatic Nerve Injury.

    PubMed

    Zhang, Weidong; Zhu, Xudong; Liu, Yang; Chen, Minhao; Yan, Shixian; Mao, Xingxing; Liu, Zhongbing; Wu, Weijie; Chen, Chen; Xu, Xinbao; Wang, Youhua

    2015-09-01

    Nur77, together with Nurr1 and NOR-1, constitutes the NR4A subgroup of orphan nuclear receptors and plays critical roles in cell proliferation, differentiation, migration, and apoptosis. Among them, Nur77 is universally well known to contribute to neurite outgrowth. However, information regarding its regulation and possible function in the peripheral nervous system is still limited. In this study, we performed a sciatic nerve injury model in adult rats and detected an increased expression of Nur77 in the sciatic nerve, which was similar to the expression of Oct-6. Immunofluorescence indicated that Nur77 was located in both axons and Schwann cells. In vitro, we observed enhanced expression of Nur77 during the process of both basic fibroblast growth factor (bFGF)-induced Schwann cells differentiation and nerve growth factor (NGF)-induced PC12 cell neurite outgrowth. In vitro and in vivo experiments indicated that inhibiting the function of Nur77 by specific short hairpin RNA could depress Schwann cells myelinization and axons regeneration. Collectively, all these results suggested that upregulation of Nur77 might be involved in Schwann cells differentiation and neurite elongation following sciatic nerve crush. PMID:25957997

  1. Neurite development in PC12 cells cultured on nanopillars and nanopores with sizes comparable with filopodia

    PubMed Central

    Haq, Furqan; Anandan, Venkatramani; Keith, Charles; Zhang, Guigen

    2007-01-01

    We investigated the effect of nanoscale topography on neurite development in pheochromocytoma (PC12 cells) by culturing the cells on substrates having nanoscale pillars and pores with sizes comparable with filipodia. We found that cells on nanopillars and nanopores developed fewer and shorter neurites than cells on smooth substrates, and that cells on nanopores developed more and longer neurites than cells on nanopillars. These results suggest that PC12 cells were spatially aware of the difference in the nanoscale structures of the underlying substrates and responded differently in their neurite extension. This finding points to the possibility of using nanoscale topographic features to control neurite development in neurons. PMID:17722518

  2. Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons.

    PubMed

    Huang, Liang; Liu, Li-Feng; Liu, Juan; Dou, Ling; Wang, Ge-Ying; Liu, Xiao-Qing; Yuan, Qiong-Lan

    2016-02-01

    Ginsenoside Rg1 (Rg1) has anti-aging and anti-neurodegenerative effects. However, the mechanisms underlying these actions remain unclear. The aim of the present study was to determine whether Rg1 affects hippocampal survival and neurite outgrowth in vitro after exposure to amyloid-beta peptide fragment 25-35 (Aβ25-35), and to explore whether the extracellular signal-regulated kinase (ERK) and Akt signaling pathways are involved in these biological processes. We cultured hippocampal neurons from newborn rats for 24 hours, then added Rg1 to the medium for another 24 hours, with or without pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) family or Akt signaling pathways for a further 24 hours. We then immunostained the neurons for growth associated protein-43, and measured neurite length. In a separate experiment, we exposed cultured hippocampal neurons to Aβ25-35 for 30 minutes, before adding Rg1 for 48 hours, with or without Akt or MAPK inhibitors, and assessed neuronal survival using Hoechst 33258 staining, and phosphorylation of ERK1/2 and Akt by western blot analysis. Rg1 induced neurite outgrowth, and this effect was blocked by API-2 (Akt inhibitor) and PD98059 (MAPK/ERK kinase inhibitor), but not by SP600125 or SB203580 (inhibitors of c-Jun N-terminal kinase and p38 MAPK, respectively). Consistent with this effect, Rg1 upregulated the phosphorylation of Akt and ERK1/2; these effects were reversed by API-2 and PD98059, respectively. In addition, Rg1 significantly reversed Aβ25-35-induced apoptosis; this effect was blocked by API-2 and PD98059, but not by SP600125 or SB203580. Finally, Rg1 significantly reversed the Aβ25-35-induced decrease in Akt and ERK1/2 phosphorylation, but API-2 prevented this reversal. Our results indicate that Rg1 enhances neurite outgrowth and protects against Aβ25-35-induced damage, and that its mechanism may involve the activation of Akt and ERK1/2 signaling. PMID:27073387

  3. Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons

    PubMed Central

    Huang, Liang; Liu, Li-feng; Liu, Juan; Dou, Ling; Wang, Ge-ying; Liu, Xiao-qing; Yuan, Qiong-lan

    2016-01-01

    Ginsenoside Rg1 (Rg1) has anti-aging and anti-neurodegenerative effects. However, the mechanisms underlying these actions remain unclear. The aim of the present study was to determine whether Rg1 affects hippocampal survival and neurite outgrowth in vitro after exposure to amyloid-beta peptide fragment 25–35 (Aβ25–35), and to explore whether the extracellular signal-regulated kinase (ERK) and Akt signaling pathways are involved in these biological processes. We cultured hippocampal neurons from newborn rats for 24 hours, then added Rg1 to the medium for another 24 hours, with or without pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) family or Akt signaling pathways for a further 24 hours. We then immunostained the neurons for growth associated protein-43, and measured neurite length. In a separate experiment, we exposed cultured hippocampal neurons to Aβ25–35 for 30 minutes, before adding Rg1 for 48 hours, with or without Akt or MAPK inhibitors, and assessed neuronal survival using Hoechst 33258 staining, and phosphorylation of ERK1/2 and Akt by western blot analysis. Rg1 induced neurite outgrowth, and this effect was blocked by API-2 (Akt inhibitor) and PD98059 (MAPK/ERK kinase inhibitor), but not by SP600125 or SB203580 (inhibitors of c-Jun N-terminal kinase and p38 MAPK, respectively). Consistent with this effect, Rg1 upregulated the phosphorylation of Akt and ERK1/2; these effects were reversed by API-2 and PD98059, respectively. In addition, Rg1 significantly reversed Aβ25–35-induced apoptosis; this effect was blocked by API-2 and PD98059, but not by SP600125 or SB203580. Finally, Rg1 significantly reversed the Aβ25–35-induced decrease in Akt and ERK1/2 phosphorylation, but API-2 prevented this reversal. Our results indicate that Rg1 enhances neurite outgrowth and protects against Aβ25–35-induced damage, and that its mechanism may involve the activation of Akt and ERK1/2 signaling. PMID:27073387

  4. Electrical Stimulation of Schwann Cells Promotes Sustained Increases in Neurite Outgrowth

    PubMed Central

    Koppes, Abigail N.; Nordberg, Andrea L.; Paolillo, Gina M.; Goodsell, Nicole M.; Darwish, Haley A.; Zhang, Linxia

    2014-01-01

    Endogenous electric fields are instructive during embryogenesis by acting to direct cell migration, and postnatally, they can promote axonal growth after injury (McCaig 1991, Al-Majed 2000). However, the mechanisms for these changes are not well understood. Application of an appropriate electrical stimulus may increase the rate and success of nerve repair by directly promoting axonal growth. Previously, DC electrical stimulation at 50 mV/mm (1 mA, 8 h duration) was shown to promote neurite outgrowth and a more pronounced effect was observed if both peripheral glia (Schwann cells) and neurons were co-stimulated. If electrical stimulation is delivered to an injury site, both the neurons and all resident non-neuronal cells [e.g., Schwann cells, endothelial cells, fibroblasts] will be treated and this biophysical stimuli can influence axonal growth directly or indirectly via changes to the resident, non-neuronal cells. In this work, non-neuronal cells were electrically stimulated, and changes in morphology and neuro-supportive cells were evaluated. Schwann cell response (morphology and orientation) was examined after an 8 h stimulation over a range of DC fields (0–200 mV/mm, DC 1 mA), and changes in orientation were observed. Electrically prestimulating Schwann cells (50 mV/mm) promoted 30% more neurite outgrowth relative to co-stimulating both Schwann cells with neurons, suggesting that electrical stimulation modifies Schwann cell phenotype. Conditioned medium from the electrically prestimulated Schwann cells promoted a 20% increase in total neurite outgrowth and was sustained for 72 h poststimulation. An 11-fold increase in nerve growth factor but not brain-derived neurotrophic factor or glial-derived growth factor was found in the electrically prestimulated Schwann cell-conditioned medium. No significant changes in fibroblast or endothelial morphology and neuro-supportive behavior were observed poststimulation. Electrical stimulation is widely used in

  5. Sargaquinoic acid promotes neurite outgrowth via protein kinase A and MAP kinases-mediated signaling pathways in PC12D cells.

    PubMed

    Kamei, Yuto; Tsang, Chi Kwan

    2003-08-01

    We previously isolated a nerve growth factor (NGF)-dependent neurite outgrowth promoting substance MC14 (sargaquinoic acid) from a marine brown alga, Sargassum macrocarpum. In the present study, the NGF-potentiating activity of MC14 to neural differentiation of PC12D cells was investigated in detail. The treatment of cells with 3 microg/ml MC14 in the presence of 1.25-100 ng/ml NGF markedly enhanced the proportion of neurite-bearing cells compared with the NGF-only controls. In addition, MC14 significantly elevated the NGF-induced specific acetylcholinesterase (AchE) activity in PC12D cells, suggesting that MC14 could morphologically and biochemically promote the differentiation of PC12D cells. The mechanism of action of MC14 was further investigated by pharmacological inhibition of several intracellular signaling molecules. Results indicated that the neurite outgrowth promoting activity of MC14 was almost completely blocked by 10 microM PD98059, suggesting that a TrkA-dependent MAP kinases-mediated signaling pathway may play a crucial role in modulating the effect of MC14. Besides, the MC14-enhanced neurite outgrowth was substantially suppressed by the pretreatment with 10 ng/ml protein kinase A (PKA) inhibitor, demonstrating that the adenylate cyclase-PKA signaling cascade was also involved in the action of MC14. In contrast, a PKC inhibitor chelerythrine chloride did not inhibit the neurite outgrowth promoting activity of MC14. Altogether, these results demonstrate that MC14 enhances the neurite outgrowth by cooperating at least two separated signaling pathways, a TrkA-MAP kinases pathway and an adenylate cyclase-PKA pathway, in PC12D cells. PMID:12850058

  6. Neurite-promoting factor in conditioned medium from RN22 Schwannoma cultures: bioassay, fractionation, and properties.

    PubMed

    Manthorpe, M; Varon, S; Adler, R

    1981-09-01

    On polyornithine (PORN) substrata dissociated 8-day chick embryo ciliary ganglionic neurons will survive if the culture medium is supplemented with Ciliary neuronotrophic Factor. However, neuritic growth will not occur unless the substratum is derivatized with a PORN-bindable Neurite Promoting Factor (PNPF). In this preliminary study we report that soluble PNPF can be (1) assayed by a convenient in vitro system; (2) obtained in relatively large amounts from serum-free media conditioned over RN22 Schwannoma cultures; (3) concentrated by using Amicon XM100 ultrafiltration; and (4) separated from nearly all of the non-active protein by using ion-exchange chromatography. The partially purified PNPF can be concentrated using XM100 and is heat- and protease-sensitive. In the course of these fractionation studies we observed in some cases a concentration-dependent interference with the expression of PNPF activity in the bioassay; we propose graphical methods to permit the simultaneous determination of PNPF and the extent of such interference. Different treatments that affected the interference property did not always affect PNPF activity in a reciprocal manner, leaving open the possibility that the interference with PNPF activity results from reversible alteration of the PNPF molecule, or that there exists a separate interfering agent. PMID:7276956

  7. Protective Effects of Rosa damascena and Its Active Constituent on Aβ(25-35)-Induced Neuritic Atrophy.

    PubMed

    Awale, Suresh; Tohda, Chihiro; Tezuka, Yasuhiro; Miyazaki, Makoto; Kadota, Shigetoshi

    2011-01-01

    Dementia is a clinical syndrome characterized by multiple cognitive deficits and causes progressive neurodegeneration leading eventually to death. The incidence of dementia is increasing worldwide with the increase in ageing population. However, no effective treatment is available yet. It has been hypothesized that drugs activating neurite outgrowth might induce neuronal reconstruction and help in the recovery of brain function. Working on this hypothesis, we recently observed that the chloroform extract of the Rosa damascena significantly induced the neurite outgrowth activity and inhibited the Aβ(25-35)-induced atrophy and cell death. Further workup led the isolation of a very long polyunsaturated fatty acid having molecular formula C(37)H(64)O(2) as an active constituent. The structure of this compound was established by extensive analysis of fragmentations observed in EI-MS mode. The isolated compound protected Aβ(25-35)-induced atrophy and displayed strong neurite outgrowth activity. The length of dendrite in the cells treated with this compound were comparable to those of nerve growth factor (NGF) treated cells. PMID:19789212

  8. TDP-43 binds and transports G-quadruplex-containing mRNAs into neurites for local translation.

    PubMed

    Ishiguro, Akira; Kimura, Nobuyuki; Watanabe, Yuto; Watanabe, Sumiko; Ishihama, Akira

    2016-05-01

    Growth and differentiation of the neurites depends on long-distance transport of a specific set of mRNAs to restricted area and their local translation. Here, we found that a TAR DNA-binding protein of 43 kDa in size (TDP-43) plays an essential role in intracellular transport of mRNA. For identification of target RNAs recognized by TDP-43, we purified TDP-43 in soluble dimer form and subjected to in vitro systematic evolution of ligands by exponential enrichment (SELEX) screening. All the TDP-43-bound RNAs were found to contain G-quadruplex (G4). Using a double-fluorescent probe system, G4-containing RNAs were found to be transported, together with TDP-43, into the distal neurites. Two lines of evidence indicated that loss of function of TDP-43 results in the neurodegenerative disorder: (i) amyotrophic lateral sclerosis (ALS)-linked mutant TDP-43M337V lacks the activity of binding and transport of G4-containing mRNAs; and (ii) RNA containing G4-forming GGGGCC repeat expansion from the ALS-linked C9orf72 gene absorbs TDP-43, thereby reducing the intracellular pool of functional TDP-43. Taken together, we propose that TDP-43 within neurons plays an essential role of mRNA transport into distal neurites for local translation, and thus, dysfunctions of TDP-43 cause neural diseases such as ALS and frontotemporal lobar degeneration. PMID:26915990

  9. Neurite outgrowth is significantly increased by the simultaneous presentation of Schwann cells and moderate exogenous electric fields

    NASA Astrophysics Data System (ADS)

    Koppes, Abigail N.; Seggio, Angela M.; Thompson, Deanna M.

    2011-08-01

    Axonal extension is influenced by a variety of external guidance cues; therefore, the development and optimization of a multi-faceted approach is probably necessary to address the intricacy of functional regeneration following nerve injury. In this study, primary dissociated neonatal rat dorsal root ganglia neurons and Schwann cells were examined in response to an 8 h dc electrical stimulation (0-100 mV mm-1). Stimulated samples were then fixed immediately, immunostained, imaged and analyzed to determine Schwann cell orientation and characterize neurite outgrowth relative to electric field strength and direction. Results indicate that Schwann cells are viable following electrical stimulation with 10-100 mV mm-1, and retain a normal morphology relative to unstimulated cells; however, no directional bias is observed. Neurite outgrowth was significantly enhanced by twofold following exposure to either a 50 mV mm-1 electric field (EF) or co-culture with unstimulated Schwann cells by comparison to neurons cultured alone. Neurite outgrowth was further increased in the presence of simultaneously applied cues (Schwann cells + 50 mV mm-1 dc EF), exhibiting a 3.2-fold increase over unstimulated control neurons, and a 1.2-fold increase over either neurons cultured with unstimulated Schwann cells or the electrical stimulus alone. These results indicate that dc electric stimulation in combination with Schwann cells may provide synergistic guidance cues for improved axonal growth relevant to nerve injuries in the peripheral nervous system.

  10. Kihi-to, a herbal traditional medicine, improves Abeta(25–35)-induced memory impairment and losses of neurites and synapses

    PubMed Central

    Tohda, Chihiro; Naito, Rie; Joyashiki, Eri

    2008-01-01

    Background We previously hypothesized that achievement of recovery of brain function after the injury requires the reconstruction of neuronal networks, including neurite regeneration and synapse reformation. Kihi-to is composed of twelve crude drugs, some of which have already been shown to possess neurite extension properties in our previous studies. The effect of Kihi-to on memory deficit has not been examined. Thus, the goal of the present study is to determine the in vivo and in vitro effects of Kihi-to on memory, neurite growth and synapse reconstruction. Methods Effects of Kihi-to, a traditional Japanese-Chinese traditional medicine, on memory deficits and losses of neurites and synapses were examined using Alzheimer's disease model mice. Improvements of Aβ(25–35)-induced neuritic atrophy by Kihi-to and the mechanism were investigated in cultured cortical neurons. Results Administration of Kihi-to for consecutive 3 days resulted in marked improvements of Aβ(25–35)-induced impairments in memory acquisition, memory retention, and object recognition memory in mice. Immunohistochemical comparisons suggested that Kihi-to attenuated neuritic, synaptic and myelin losses in the cerebral cortex, hippocampus and striatum. Kihi-to also attenuated the calpain increase in the cerebral cortex and hippocampus. When Kihi-to was added to cells 4 days after Aβ(25–35) treatment, axonal and dendritic outgrowths in cultured cortical neurons were restored as demonstrated by extended lengths of phosphorylated neurofilament-H (P-NF-H) and microtubule-associated protein (MAP)2-positive neurites. Aβ(25–35)-induced cell death in cortical culture was also markedly inhibited by Kihi-to. Since NF-H, MAP2 and myelin basic protein (MBP) are substrates of calpain, and calpain is known to be involved in Aβ-induced axonal atrophy, expression levels of calpain and calpastatin were measured. Treatment with Kihi-to inhibited the Aβ(25–35)-evoked increase in the calpain level and

  11. Human Umbilical Tissue-Derived Cells Promote Synapse Formation and Neurite Outgrowth via Thrombospondin Family Proteins

    PubMed Central

    Koh, Sehwon; Kim, Namsoo; Yin, Henry H.; Harris, Ian R.; Dejneka, Nadine S.

    2015-01-01

    Cell therapy demonstrates great potential for the treatment of neurological disorders. Human umbilical tissue-derived cells (hUTCs) were previously shown to have protective and regenerative effects in animal models of stroke and retinal degeneration, but the underlying therapeutic mechanisms are unknown. Because synaptic dysfunction, synapse loss, degeneration of neuronal processes, and neuronal death are hallmarks of neurological diseases and retinal degenerations, we tested whether hUTCs contribute to tissue repair and regeneration by stimulating synapse formation, neurite outgrowth, and neuronal survival. To do so, we used a purified rat retinal ganglion cell culture system and found that hUTCs secrete factors that strongly promote excitatory synaptic connectivity and enhance neuronal survival. Additionally, we demonstrated that hUTCs support neurite outgrowth under normal culture conditions and in the presence of the growth-inhibitory proteins chondroitin sulfate proteoglycan, myelin basic protein, or Nogo-A (reticulon 4). Furthermore, through biochemical fractionation and pharmacology, we identified the major hUTC-secreted synaptogenic factors as the thrombospondin family proteins (TSPs), TSP1, TSP2, and TSP4. Silencing TSP expression in hUTCs, using small RNA interference, eliminated both the synaptogenic function of these cells and their ability to promote neurite outgrowth. However, the majority of the prosurvival functions of hUTC-conditioned media was spared after TSP knockdown, indicating that hUTCs secrete additional neurotrophic factors. Together, our findings demonstrate that hUTCs affect multiple aspects of neuronal health and connectivity through secreted factors, and each of these paracrine effects may individually contribute to the therapeutic function of these cells. SIGNIFICANCE STATEMENT Human umbilical tissue-derived cells (hUTC) are currently under clinical investigation for the treatment of geographic atrophy secondary to age-related macular

  12. Automated quantification of neurite outgrowth orientation distributions on patterned surfaces

    NASA Astrophysics Data System (ADS)

    Payne, Matthew; Wang, Dadong; Sinclair, Catriona M.; Kapsa, Robert M. I.; Quigley, Anita F.; Wallace, Gordon G.; Razal, Joselito M.; Baughman, Ray H.; Münch, Gerald; Vallotton, Pascal

    2014-08-01

    Objective. We have developed an image analysis methodology for quantifying the anisotropy of neuronal projections on patterned substrates. Approach. Our method is based on the fitting of smoothing splines to the digital traces produced using a non-maximum suppression technique. This enables precise estimates of the local tangents uniformly along the neurite length, and leads to unbiased orientation distributions suitable for objectively assessing the anisotropy induced by tailored surfaces. Main results. In our application, we demonstrate that carbon nanotubes arrayed in parallel bundles over gold surfaces induce a considerable neurite anisotropy; a result which is relevant for regenerative medicine. Significance. Our pipeline is generally applicable to the study of fibrous materials on 2D surfaces and should also find applications in the study of DNA, microtubules, and other polymeric materials.

  13. Roles of actin filaments and three second-messenger systems in short-term regulation of chick dorsal root ganglion neurite outgrowth.

    PubMed

    Lankford, K L; Letourneau, P C

    1991-01-01

    In a previous study (J. Cell Biol. 109: 1229-1243, 1989), we reported that conditions which increased growth cone calcium levels and induced neurite retraction in cultured chick DRG neurons also resulted in an apparent loss of actin filaments in the growth cone periphery. We further showed that the actin-stabilizing drug phalloidin could block or reverse calcium-ionophore-induced neurite retraction, indicating that the behavioral changes were mediated, at least in part, by changes in actin filament stability. In this study, we have further characterized the calcium sensitivity of growth cone behavior to identify which features of calcium-induced behavioral effects can be attributed to effects on actin filaments alone, and to assess whether two other second-messenger systems, cAMP and protein kinase C, might influence neurite outgrowth by altering calcium levels or actin stability. The results indicated that growth cone behavior was highly sensitive to small changes in calcium concentrations. Neurite outgrowth was only observed in calcium-permeabilized cells when extracellular calcium concentrations were between 200 and 300 nM, and changes as small as 50 nM commonly produced detectable changes in behavior. Furthermore, low doses of cytochalasins mimicked all of the grossly observable features of growth cone responses to elevation of intracellular calcium, including the apparent preferential destruction of lamellipodial actin filaments and sparing of filopodial actin, suggesting that the behavioral effects of calcium elevation could be explained by loss of actin filaments alone. The effects of cAMP elevation and protein kinase C activation on growth cone behavior, ultrastructure, and fura2-AM-measured calcium levels indicated that the effects of cAMP manipulations could be partially explained by a cAMP-induced lowering of growth cone calcium levels and concomitant increased stabilization of actin filaments, but protein kinase C appeared to act through an independent

  14. Metastasis suppressor 1 regulates neurite outgrowth in primary neuron cultures.

    PubMed

    Yu, Juan; Lin, Shuyun; Wang, Mei; Liang, Lijun; Zou, Zijiao; Zhou, Xinfeng; Wang, Meichi; Chen, Ping; Wang, Ying

    2016-10-01

    Metastasis suppressor 1 (MTSS1) or missing in metastasis (MIM) is an actin- and membrane-binding protein with tumor suppressor functions. MTSS1 is important for cell morphology, motility, metastasis. The role of MTSS1 in cell morphology has been widely investigated in non-neuronal tissues; however the role of MTSS1 in neurite outgrowth remains unclear. Here we investigated the effect of MTSS1 on neurite outgrowth in primary cerebellar granule and hippocampal neurons of mouse. We found that overexpression of MTSS1 in cerebellar granule neurons significantly enhanced dendrite elaboration but inhibited axon elongation. This phenotype was significantly reduced by deletion of the Wiskott-Aldrich homology 2 (WH2) motif and point mutation in the insulin receptor substrate p53 (IRSp53) and MIM/MTSS1 homology (IMD) domain. Furthermore, inhibition of Rac1 activity or blocking of phosphatidyl inositol phosphates (PIPs) signaling decreased the effect of MTSS1 markedly. In accordance with the over-expression data, knockdown of MTSS1 in cerebellar granule neurons could increase the axon length but decrease the dendrite length and the number of dendrites. In addition, MTSS1 knock down in embryonic hippocampal neurons suppressed neurite branching and reduced dendrite length. Our findings have demonstrated that MTSS1 modulates neuronal morphology, possibly through a Rac1-PIPs signaling pathway. PMID:27401056

  15. Neurite outgrowth on fluorinated polyimide film micropatterned by ion irradiation

    NASA Astrophysics Data System (ADS)

    Okuyama, Y.; Sato, M.; Nagaoka, S.; Kawakami, H.; Suzuki, Y.; Iwaki, M.

    2003-05-01

    In this study, we investigated neurite outgrowth on a fluorinated polyimide film micropatterned by ion irradiation. We used the fluorinated polyimide because of its excellent thermal and mechanical properties and biocompatibility. Rattus norvegicus chromaphin (PC12) cells were used for in vitro studies. The polyimide films were irradiated with He +, Ne + or Kr + at 1 × 10 14 ions/cm 2 using an ion-beam mask. The lines in the mask were 120 and 160 μm wide and 120-160 μm apart. PC12 cells were selectively adhered on the polyimide film micropatterned by Kr +-irradiation. However, the neurite length on the film irradiated by Kr + was shorter than that determined in the film irradiated by He +. On the other hand, neurite outgrowth on the polyimide film micropatterned by He +-irradiation was at least 100 μm in length. This initial study indicated the enhanced outgrowth of PC12 cells on the fluorinated polyimide film micropatterned by ion irradiation.

  16. Stimulation of neurite outgrowth using positively charged hydrogels

    PubMed Central

    Dadsetan, Mahrokh; Knight, Andrew M.; Lu, Lichun; Windebank, Anthony J.; Yaszemski, Michael J.

    2009-01-01

    Autologous nerve grafts are currently the best option for the treatment of segmental peripheral nerve defects. However, autografts have several drawbacks including size mismatch and loss of sensation in the donor nerve’s sensory distribution. In this work, we have investigated the development of a synthetic hydrogel that contains positive charge for use as a substrate for nerve cell attachment and neurite outgrowth in culture. We have demonstrated that modification of oligo-(polyethylene glycol) fumarate (OPF) with a positively charged monomer improves primary sensory rat neuron attachment and differentiation in a dose-dependent manner. Positively charged hydrogels also supported attachment of dorsal root ganglion (DRG) explants that contain sensory neurons, Schwann cells and neuronal support cells. Furthermore, charged hydrogels were analyzed for the appearance of myelinated structures in a co-culture containing DRG neurons and Schwann cells. DRGs and Schwann cells remained viable on charged hydrogels for a time period of three weeks and neurites extended from the DRGs. Sudan black staining revealed that neurites emerging from DRGs were accompanied by migrating Schwann cells. These findings suggest that charged OPF hydrogels are capable of sustaining both primary nerve cells and the neural support cells that are critical for regeneration. PMID:19427689

  17. The type I inositol 1,4,5-trisphosphate receptor interacts with protein 4.1N to mediate neurite formation through intracellular Ca waves.

    PubMed

    Fiedler, Michael J; Nathanson, Michael H

    2011-01-01

    Ca(2+) waves are an important mechanism for encoding Ca(2+) signaling information, but the molecular basis for wave formation and how this regulates neuronal function is not entirely understood. Using nerve growth factor-differentiated PC12 cells as a model system, we investigated the interaction between the type I inositol 1,4,5-trisphosphate receptor (IP3R1) and the cytoskeletal linker, protein 4.1N, to examine the relationship between Ca(2+) wave formation and neurite development. This was examined using RNAi and overexpressed dominant negative binding regions of each protein. Confocal microscopy was used to monitor neurite formation and Ca(2+) waves. Knockdown of IP3R1 or 4.1N attenuated neurite formation, as did binding regions of IP3R1 and 4.1N, which colocalized with endogenous 4.1N and IP3R1, respectively. Upon stimulation with the IP3-producing agonist carbachol, both RNAi and dominant negative molecules shifted signaling events from waves to homogeneous patterns of Ca(2+) release. These findings provide evidence that IP3R1 localization, via protein 4.1N, is necessary for Ca(2+) wave formation, which in turn mediates neurite formation. PMID:21389686

  18. The Type I Inositol 1,4,5-Trisphosphate Receptor Interacts with Protein 4.1N to Mediate Neurite Formation through Intracellular Ca2+ Waves

    PubMed Central

    Fiedler, Michael J.; Nathanson, Michael H.

    2011-01-01

    Ca2+ waves are an important mechanism for encoding Ca2+ signaling information, but the molecular basis for wave formation and how this regulates neuronal function is not entirely understood. Using nerve growth factor-differentiated PC12 cells as a model system, we investigated the interaction between the type I inositol 1,4,5-trisphosphate receptor (IP3R1) and the cytoskeletal linker, protein 4.1N, to examine the relationship between Ca2+ wave formation and neurite development. This was examined using RNAi and overexpressed dominant negative binding regions of each protein. Confocal microscopy was used to monitor neurite formation and Ca2+ waves. Knockdown of IP3R1 or 4.1N attenuated neurite formation, as did binding regions of IP3R1 and 4.1N, which colocalized with endogenous 4.1N and IP3R1, respectively. Upon stimulation with the IP3-producing agonist carbachol, both RNAi and dominant negative molecules shifted signaling events from waves to homogeneous patterns of Ca2+ release. These findings provide evidence that IP3R1 localization, via protein 4.1N, is necessary for Ca2+ wave formation, which in turn mediates neurite formation. PMID:21389686

  19. Differentiated adipose-derived stem cells act synergistically with RGD-modified surfaces to improve neurite outgrowth in a co-culture model.

    PubMed

    de Luca, A C; Faroni, A; Downes, S; Terenghi, G

    2016-08-01

    Peripheral nerve damage is a problem encountered after trauma and during surgery and the development of synthetic polymer conduits may offer a promising alternative to autografts. In order to improve the performance of the polymer to be used for nerve conduits, poly-ε-caprolactone (PCL) films were chemically functionalized with RGD moieties, using a chemical reaction previously developed. In vitro cultures of dissociated dorsal root ganglion (DRG) neurons provide a valid model to study different factors affecting axonal growth. In this work, DRG neurons were cultured on RGD-functionalized PCL films. Adult adipose-derived stem cells differentiated to Schwann cells (dASCs) were initially cultured on the functionalized PCL films, resulting in improved attachment and proliferation. dASCs were also co-cultured with DRG neurons on treated and untreated PCL to assess stimulation by dASCs on neurite outgrowth. Neuron response was generally poor on untreated PCL films, but long neurites were observed in the presence of dASCs or RGD moieties. A combination of the two factors enhanced even further neurite outgrowth, acting synergistically. Finally, in order to better understand the extracellular matrix (ECM)-cell interaction, a β1 integrin blocking experiment was carried out. Neurite outgrowth was not affected by the specific antibody blocking, showing that β1 integrin function can be compensated by other molecules present on the cell membrane. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23950058

  20. The conditioning lesion effect on sympathetic neurite outgrowth is dependent on gp130 cytokines

    PubMed Central

    Sachs, H. Hyatt; Rohrer, H.; Zigmond, R.E.

    2010-01-01

    Sympathetic neurons, like sensory neurons, increase neurite outgrowth after a conditioning lesion. Studies in leukemia inhibitory factor (LIF) knockout animals showed that the conditioning lesion effect in sensory neurons is dependent in part on this cytokine; however, similar studies on sympathetic neurons revealed no such effect. Comparable studies with sensory neurons taken from mice lacking the related cytokine interleukin-6 (IL-6) have yielded conflicting results. LIF and IL-6 belong to a family of cytokines known as the gp130 family because they act on receptors containing the subunit gp130. In sympathetic ganglia, axotomy leads to increases in mRNA for four of these cytokines (LIF, IL-6, IL-11, and on-costatin M). To test the role of this family of cytokines as a whole in the conditioning lesion response in sympathetic neurons, mice in which gp130 was selectively eliminated in noradrenergic neurons were studied. The postganglionic axons of the SCG were transected, and seven days later the ganglia were removed and neurite outgrowth was measured in explant and dissociated cell cultures. In both systems, neurons from wild type animals showed enhanced growth after a conditioning lesion. In contrast, no enhancement occurred in neurons from mutant animals. This lack of stimulation of outgrowth occurred despite an increase in expression of activating transcription factor 3 (ATF3) in the mutant mice. These studies demonstrate that stimulation of enhanced growth of sympathetic neurons after a conditioning lesion is dependent on gp130 cytokine signaling and is blocked in the absence of signaling by these cytokines in spite of an increase in ATF3. PMID:20144891

  1. ProNGF derived from rat sciatic nerves downregulates neurite elongation and axon specification in PC12 cells

    PubMed Central

    Trigos, Anna Sofía; Longart, Marines; García, Lisbeth; Castillo, Cecilia; Forsyth, Patricia; Medina, Rafael

    2015-01-01

    Several reports have shown that a sciatic nerve conditioned media (CM) causes neuronal-like differentiation in PC12 cells. This differentiation is featured by neurite outgrowth, which are exclusively dendrites, without axon or sodium current induction. In previous studies, our group reported that the CM supplemented with a generic inhibitor for tyrosine kinase receptors (k252a) enhanced the CM-induced morphological differentiation upregulating neurite outgrowth, axonal formation and sodium current elicitation. Sodium currents were also induced by depletion of endogenous precursor of nerve growth factorr (proNGF) from the CM (pNGFd-CM). Given that sodium currents, neurite outgrowth and axon specification are important features of neuronal differentiation, in the current manuscript, first we investigated if proNGF was hindering the full PC12 cell neuronal-like differentiation. Second, we studied the effects of exogenous wild type (pNGFwt) and mutated (pNGFmut) proNGF isoforms over sodium currents and whether or not their addition to the pNGFd-CM would prevent sodium current elicitation. Third, we investigated if proNGF was exerting its negative regulation through the sortilin receptor, and for this, the proNGF action was blocked with neurotensin (NT), a factor known to compete with proNGF for sortilin. Thereby, here we show that pNGFd-CM enhanced cell differentiation, cell proportion with long neurites, total neurite length, induced axonal formation and sodium current elicitation. Interestingly, treatment of PC12 cells with wild type or mutated proNGF isoforms elicited sodium currents. Supplementing pNGFd-CM with pNGFmut reduced 35% the sodium currents. On the other hand, pNGFd-CM+pNGFwt induced larger sodium currents than pNGFd-CM. Finally, treatments with CM supplemented with NT showed that sortilin was mediating proNGF negative regulation, since its blocking induced similar effects than the pNGFd-CM treatment. Altogether, our results suggest that proNGF within the

  2. Iridoids and sesquiterpenoids of Valeriana stenoptera and their effects on NGF-induced neurite outgrowth in PC12 cells.

    PubMed

    Dong, Fa-Wu; Wu, Zhi-Kun; Yang, Liu; Zi, Chen-Ting; Yang, Dan; Ma, Rui-Jing; Liu, Zhen-Hua; Luo, Huai-Rong; Zhou, Jun; Hu, Jiang-Miao

    2015-10-01

    Twenty-one compounds (nine iridoids and twelve sesquiterpenoids), including ten previously unknown (five iridoids and five sesquiterpenoids) were isolated from whole dried material of Valeriana stenoptera. Structures were established on the basis of extensive spectroscopic analysis and the relative stereochemistry of 13-hydroxypatchoulol A was further confirmed by X-ray crystallographic data. All isolates were evaluated for their effects on nerve growth factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells and seven compounds showed potent promoting effects. PMID:26343415

  3. Nebulized solvent ablation of aligned PLLA fibers for the study of neurite response to anisotropic-to-isotropic fiber/film transition (AFFT) boundaries in astrocyte–neuron co-cultures

    PubMed Central

    Zuidema, Jonathan M.; Desmond, Gregory P.; Rivet, Christopher J.; Kearns, Kathryn R.; Thompso, Deanna M.; Gilbert, Ryan J.

    2015-01-01

    Developing robust in vitro models of in vivo environments has the potential to reduce costs and bring new therapies from the bench top to the clinic more efficiently. This study aimed to develop a biomaterial platform capable of modeling isotropic-to-anisotropic cellular transitions observed in vivo, specifically focusing on changes in cellular organization following spinal cord injury. In order to accomplish this goal, nebulized solvent patterning of aligned, electrospun poly-l-lactic acid (PLLA) fiber substrates was developed. This method produced a clear topographic transitional boundary between aligned PLLA fibers and an isotropic PLLA film region. Astrocytes were then seeded on these scaffolds, and a shift between oriented and non-oriented astrocytes was created at the anisotropic-to-isotropic fiber/film transition (AFFT) boundary. Orientation of chondroitin sulfate proteoglycans (CSPGs) and fibronectin produced by these astrocytes was analyzed, and it was found that astrocytes growing on the aligned fibers produced aligned arrays of CSPGs and fibronectin, while astrocytes growing on the isotropic film region produced randomly-oriented CSPG and fibronectin arrays. Neurite extension from rat dissociated dorsal root ganglia (DRG) was studied on astrocytes cultured on anisotropic, aligned fibers, isotropic films, or from fibers to films. It was found that neurite extension was oriented and longer on PLLA fibers compared to PLLA films. When dissociated DRG were cultured on the astrocytes near the AFFT boundary, neurites showed directed orientation that was lost upon growth into the isotropic film region. The AFFT boundary also restricted neurite extension, limiting the extension of neurites once they grew from the fibers and into the isotropic film region. This study reveals the importance of anisotropic-to-isotropic transitions restricting neurite outgrowth by itself. Furthermore, we present this scaffold as an alternative culture system to analyze neurite

  4. GTP Hydrolysis of TC10 Promotes Neurite Outgrowth through Exocytic Fusion of Rab11- and L1-Containing Vesicles by Releasing Exocyst Component Exo70

    PubMed Central

    Fujita, Akane; Koinuma, Shingo; Yasuda, Sayaka; Nagai, Hiroyuki; Kamiguchi, Hiroyuki; Wada, Naoyuki; Nakamura, Takeshi

    2013-01-01

    The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be

  5. Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata

    PubMed Central

    Jonas, Robin; Klusch, Andreas; Schmelz, Martin; Petersen, Marlen; Carr, Richard W.

    2015-01-01

    Nine isoforms of voltage-gated sodium channels (NaV) have been characterized and in excitable tissues they are responsible for the initiation and conduction of action potentials. For primary afferent neurons residing in dorsal root ganglia (DRG), individual neurons may express multiple NaV isoforms extending the neuron’s functional capabilities. Since expression of NaV isoforms can be differentially regulated by neurotrophic factors we have examined the functional consequences of exposure to either nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) on action potential conduction in outgrowing cultured porcine neurites of DRG neurons. Calcium signals were recorded using the exogenous intensity based calcium indicator Fluo-8®, AM. In 94 neurons, calcium signals were conducted along neurites in response to electrical stimulation of the soma. At an image acquisition rate of 25 Hz it was possible to discern calcium transients in response to individual electrical stimuli. The peak amplitude of electrically-evoked calcium signals was limited by the ability of the neuron to follow the stimulus frequency. The stimulus frequency required to evoke a half-maximal calcium response was approximately 3 Hz at room temperature. In 13 of 14 (93%) NGF-responsive neurites, TTX-r NaV isoforms alone were sufficient to support propagated signals. In contrast, calcium signals mediated by TTX-r NaVs were evident in only 4 of 11 (36%) neurites from somata cultured in GDNF. This establishes a basis for assessing action potential signaling using calcium imaging techniques in individual cultured neurites and suggests that, in the pig, afferent nociceptor classes relying on the functional properties of TTX-r NaV isoforms, such as cold-nociceptors, most probably derive from NGF-responsive DRG neurons. PMID:26407014

  6. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells differentiate into a Schwann-cell phenotype and promote neurite outgrowth in vitro.

    PubMed

    Peng, Jiang; Wang, Yu; Zhang, Li; Zhao, Bin; Zhao, Zhe; Chen, JiFeng; Guo, QuanYi; Liu, ShuYun; Sui, Xiang; Xu, WenJing; Lu, ShiBi

    2011-02-28

    Cell-based therapy has achieved promising functional recovery for peripheral nerve repair. Although Schwann cells (SCs) and bone marrow derived mesenchymal stromal cells (BM-MSCs) are the main cell source for nerve tissue engineering, the clinical application is limited because of donor site morbidity, the invasive procedure, and the decreased number of SCs and BM-MSCs. Wharton's jelly-derived mesenchymal stem cells (WJMSCs) could be a promising cell source for nerve tissue engineering because they are easily accessible and their use has no ethical issues. We investigated the phenotypic, molecular and functional characteristics of WJMSCs differentiated along a Schwann-cell lineage. Cultured WJMSCs were isolated from human umbilical cord, and the undifferentiated WJMSCs were confirmed by the detection of MSC-specific cell-surface markers. WJMSCs treated with a mixture of glial growth factors (basic fibroblast growth factor, platelet-derived growth factor and forskolin) adopted a spindle-like morphology similar to SCs. Immunocytochemical staining, RT-PCR analysis, and Western blot analysis revealed that the treated cells expressed the glial markers glial fibrillary acidic protein, p75, S100 and P0 and indicative of differentiation. On co-culture with dorsal root ganglia neurons, the differentiated WJMSCs enhanced the number of sprouting neurites and neurite length in dorsal root ganglia neurons. Furthermore, using enzyme-linked immunosorbent assay and RT-PCR methodology, we found differentiated WJMSCs secrete and express neurotrophic factors, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). Quantification of neurite outgrowth from PC12 cells grown in differentiated WJMSCs-conditioned media demonstrates that the neurite length is significantly more than control medium and undifferentiated WJMSCs group. WJMSCs can be differentiated into cells that are Schwann-like in terms of morphologic features, phenotype, and

  7. Astrocytic αVβ3 Integrin Inhibits Neurite Outgrowth and Promotes Retraction of Neuronal Processes by Clustering Thy-1

    PubMed Central

    Herrera-Molina, Rodrigo; Frischknecht, Renato; Maldonado, Horacio; Seidenbecher, Constanze I.; Gundelfinger, Eckart D.; Hetz, Claudio; Aylwin, María de la Luz; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

    2012-01-01

    Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to αVβ3 integrin in trans eliciting responses in astrocytes. Nonetheless, whether αVβ3 integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of αVβ3 integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous αVβ3 integrin restricted neurite outgrowth. Likewise, αVβ3-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(−) CAD cells. In differentiating primary neurons exposed to αVβ3-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, αVβ3-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by αVβ3 integrin. Binding of αVβ3-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, αVβ3-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that αVβ3 integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage. PMID:22479590

  8. A Farnesyltransferase Acts to Inhibit Ectopic Neurite Formation in C. elegans

    PubMed Central

    Carr, David; Sanchez-Alvarez, Leticia; Imai, Janice H.; Slatculescu, Cristina; Noblett, Nathaniel; Mao, Lei; Beese, Lorena; Colavita, Antonio

    2016-01-01

    Genetic pathways that regulate nascent neurite formation play a critical role in neuronal morphogenesis. The core planar cell polarity components VANG-1/Van Gogh and PRKL-1/Prickle are involved in blocking inappropriate neurite formation in a subset of motor neurons in C. elegans. A genetic screen for mutants that display supernumerary neurites was performed to identify additional factors involved in this process. This screen identified mutations in fntb-1, the β subunit of farnesyltransferase. We show that fntb-1 is expressed in neurons and acts cell-autonomously to regulate neurite formation. Prickle proteins are known to be post-translationally modified by farnesylation at their C-terminal CAAX motifs. We show that PRKL-1 can be recruited to the plasma membrane in both a CAAX-dependent and CAAX-independent manner but that PRKL-1 can only inhibit neurite formation in a CAAX-dependent manner. PMID:27300162

  9. The neuritic plaque facilitates pathological conversion of tau in an Alzheimer's disease mouse model

    PubMed Central

    Li, Tong; Braunstein, Kerstin E.; Zhang, Juhong; Lau, Ashley; Sibener, Leslie; Deeble, Christopher; Wong, Philip C.

    2016-01-01

    A central question in Alzheimer's Disease (AD) is whether the neuritic plaque is necessary and sufficient for the development of tau pathology. Hyperphosphorylation of tau is found within dystrophic neurites surrounding β-amyloid deposits in AD mouse models but the pathological conversion of tau is absent. Likewise, expression of a human tau repeat domain in mice is insufficient to drive the pathological conversion of tau. Here we developed an Aβ-amyloidosis mouse model that expresses the human tau repeat domain and show that in these mice, the neuritic plaque facilitates the pathological conversion of wild-type tau. We show that this tau fragment seeds the neuritic plaque-dependent pathological conversion of wild-type tau that spreads from the cortex and hippocampus to the brain stem. These results establish that in addition to the neuritic plaque, a second determinant is required to drive the conversion of wild-type tau. PMID:27373369

  10. Co-effects of matrix low elasticity and aligned topography on stem cell neurogenic differentiation and rapid neurite outgrowth

    NASA Astrophysics Data System (ADS)

    Yao, Shenglian; Liu, Xi; Yu, Shukui; Wang, Xiumei; Zhang, Shuming; Wu, Qiong; Sun, Xiaodan; Mao, Haiquan

    2016-05-01

    The development of novel biomaterials that deliver precise regulatory signals to direct stem cell fate for nerve regeneration is the focus of current intensive research efforts. In this study, a hierarchically aligned fibrillar fibrin hydrogel (AFG) that was fabricated through electrospinning and the concurrent molecular self-assembly process mimics both the soft and oriented features of nerve tissue, thus providing hybrid biophysical cues to instruct cell behavior in vitro and in vivo. The electrospun hydrogels were examined by scanning electron microscopy (SEM), polarized light microscopy, small angle X-ray scattering assay and atomic force microscopy (AFM), showing a hierarchically linear-ordered structure from the nanoscale to the macroscale with a soft elastic character (elasticity ~1 kPa). We found that this low elasticity and aligned topography of AFG exhibit co-effects on promoting the neurogenic differentiation of human umbilical cord mesenchymal stem cells (hUMSCs) in comparison to random fibrin hydrogel (RFG) and tissue culture plate (TCP) control after two week cell culture in growth medium lacking supplementation with soluble neurogenic induction factors. In addition, AFG also induces dorsal root ganglion (DRG) neurons to rapidly project numerous long neurite outgrowths longitudinally along the AFG fibers for a total neurite extension distance of 1.96 mm in three days in the absence of neurotrophic factor supplementation. Moreover, the AFG implanted in a rat T9 dorsal hemisection spinal cord injury model was found to promote endogenous neural cell fast migration and axonal invasion along AFG fibers, resulting in aligned tissue cables in vivo. Our results suggest that matrix stiffness and aligned topography may instruct stem cell neurogenic differentiation and rapid neurite outgrowth, providing great promise for biomaterial design for applications in nerve regeneration.The development of novel biomaterials that deliver precise regulatory signals to

  11. PAK-PIX interactions regulate adhesion dynamics and membrane protrusion to control neurite outgrowth.

    PubMed

    Santiago-Medina, Miguel; Gregus, Kelly A; Gomez, Timothy M

    2013-03-01

    The roles of P21-activated kinase (PAK) in the regulation of axon outgrowth downstream of extracellular matrix (ECM) proteins are poorly understood. Here we show that PAK1-3 and PIX are expressed in the developing spinal cord and differentially localize to point contacts and filopodial tips within motile growth cones. Using a specific interfering peptide called PAK18, we found that axon outgrowth is robustly stimulated on laminin by partial inhibition of PAK-PIX interactions and PAK function, whereas complete inhibition of PAK function stalls axon outgrowth. Furthermore, modest inhibition of PAK-PIX stimulates the assembly and turnover of growth cone point contacts, whereas strong inhibition over-stabilizes adhesions. Point mutations within PAK confirm the importance of PIX binding. Together our data suggest that regulation of PAK-PIX interactions in growth cones controls neurite outgrowth by influencing the activity of several important mediators of actin filament polymerization and retrograde flow, as well as integrin-dependent adhesion to laminin. PMID:23321640

  12. Co-effects of matrix low elasticity and aligned topography on stem cell neurogenic differentiation and rapid neurite outgrowth.

    PubMed

    Yao, Shenglian; Liu, Xi; Yu, Shukui; Wang, Xiumei; Zhang, Shuming; Wu, Qiong; Sun, Xiaodan; Mao, Haiquan

    2016-05-21

    The development of novel biomaterials that deliver precise regulatory signals to direct stem cell fate for nerve regeneration is the focus of current intensive research efforts. In this study, a hierarchically aligned fibrillar fibrin hydrogel (AFG) that was fabricated through electrospinning and the concurrent molecular self-assembly process mimics both the soft and oriented features of nerve tissue, thus providing hybrid biophysical cues to instruct cell behavior in vitro and in vivo. The electrospun hydrogels were examined by scanning electron microscopy (SEM), polarized light microscopy, small angle X-ray scattering assay and atomic force microscopy (AFM), showing a hierarchically linear-ordered structure from the nanoscale to the macroscale with a soft elastic character (elasticity ∼1 kPa). We found that this low elasticity and aligned topography of AFG exhibit co-effects on promoting the neurogenic differentiation of human umbilical cord mesenchymal stem cells (hUMSCs) in comparison to random fibrin hydrogel (RFG) and tissue culture plate (TCP) control after two week cell culture in growth medium lacking supplementation with soluble neurogenic induction factors. In addition, AFG also induces dorsal root ganglion (DRG) neurons to rapidly project numerous long neurite outgrowths longitudinally along the AFG fibers for a total neurite extension distance of 1.96 mm in three days in the absence of neurotrophic factor supplementation. Moreover, the AFG implanted in a rat T9 dorsal hemisection spinal cord injury model was found to promote endogenous neural cell fast migration and axonal invasion along AFG fibers, resulting in aligned tissue cables in vivo. Our results suggest that matrix stiffness and aligned topography may instruct stem cell neurogenic differentiation and rapid neurite outgrowth, providing great promise for biomaterial design for applications in nerve regeneration. PMID:27124547

  13. A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

    SciTech Connect

    Sharma, Aarti; Lambrechts, Anja; Le thi Hao; Le, Thanh T.; Sewry, Caroline A.; Ampe, Christophe; Burghes, Arthur H.M.; Morris, Glenn E. . E-mail: glenn.morris@rjah.nhs.uk

    2005-09-10

    Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with {beta}-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.

  14. Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling.

    PubMed

    Fusco, Ludovico; Lefort, Riwal; Smith, Kevin; Benmansour, Fethallah; Gonzalez, German; Barillari, Caterina; Rinn, Bernd; Fleuret, Francois; Fua, Pascal; Pertz, Olivier

    2016-01-01

    Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth. PMID:26728857

  15. Triggering of high-speed neurite outgrowth using an optical microheater

    PubMed Central

    Oyama, Kotaro; Zeeb, Vadim; Kawamura, Yuki; Arai, Tomomi; Gotoh, Mizuho; Itoh, Hideki; Itabashi, Takeshi; Suzuki, Madoka; Ishiwata, Shin’ichi

    2015-01-01

    Optical microheating is a powerful non-invasive method for manipulating biological functions such as gene expression, muscle contraction, and cell excitation. Here, we demonstrate its potential usage for regulating neurite outgrowth. We found that optical microheating with a water-absorbable 1,455-nm laser beam triggers directional and explosive neurite outgrowth and branching in rat hippocampal neurons. The focused laser beam under a microscope rapidly increases the local temperature from 36 °C to 41 °C (stabilized within 2 s), resulting in the elongation of neurites by more than 10 μm within 1 min. This high-speed, persistent elongation of neurites was suppressed by inhibitors of both microtubule and actin polymerization, indicating that the thermosensitive dynamics of these cytoskeletons play crucial roles in this heat-induced neurite outgrowth. Furthermore, we showed that microheating induced the regrowth of injured neurites and the interconnection of neurites. These results demonstrate the efficacy of optical microheating methods for the construction of arbitrary neural networks. PMID:26568288

  16. Charge-balanced biphasic electrical stimulation inhibits neurite extension of spiral ganglion neurons.

    PubMed

    Shen, Na; Liang, Qiong; Liu, Yuehong; Lai, Bin; Li, Wen; Wang, Zhengmin; Li, Shufeng

    2016-06-15

    Intracochlear application of exogenous or transgenic neurotrophins, such as neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF), could promote the resprouting of spiral ganglion neuron (SGN) neurites in deafened animals. These resprouting neurites might reduce the gap between cochlear implant electrodes and their targeting SGNs, allowing for an improvement of spatial resolution of electrical stimulation. This study is to investigate the impact of electrical stimulation employed in CI on the extension of resprouting SGN neurites. We established an in vitro model including the devices delivering charge-balanced biphasic electrical stimulation, and spiral ganglion (SG) dissociated culture treated with BDNF and NT-3. After electrical stimulation with varying durations and intensities, we quantified neurite lengths and Schwann cell densities in SG cultures. Stimulations that were greater than 50μA or longer than 8h significantly decreased SG neurite length. Schwann cell density under 100μA electrical stimulation for 48h was significantly lower compared to that in non-stimulated group. These electrical stimulation-induced decreases of neurite extension and Schwann cell density were attenuated by various types of voltage-dependent calcium channel (VDCC) blockers, or completely prevented by their combination, cadmium or calcium-free medium. Our study suggested that charge-balanced biphasic electrical stimulation inhibited the extension of resprouting SGN neurites and decreased Schwann cell density in vitro. Calcium influx through multiple types of VDCCs was involved in the electrical stimulation-induced inhibition. PMID:27163199

  17. Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

    PubMed Central

    Fusco, Ludovico; Lefort, Riwal; Smith, Kevin; Benmansour, Fethallah; Gonzalez, German; Barillari, Caterina; Rinn, Bernd; Fleuret, Francois; Fua, Pascal

    2016-01-01

    Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth. PMID:26728857

  18. Neurite outgrowth stimulatory effects of myco synthesized AuNPs from Hericium erinaceus (Bull.: Fr.) Pers. on pheochromocytoma (PC-12) cells

    PubMed Central

    Raman, Jegadeesh; Lakshmanan, Hariprasath; John, Priscilla A; Zhijian, Chan; Periasamy, Vengadesh; David, Pamela; Naidu, Murali; Sabaratnam, Vikineswary

    2015-01-01

    Background Hericium erinaceus has been reported to have a wide range of medicinal properties such as stimulation of neurite outgrowth, promotion of functional recovery of axonotmetic peroneal nerve injury, antioxidant, antihypertensive, and antidiabetic properties. In recent years, the green synthesis of gold nanoparticles (AuNPs) has attracted intense interest due to the potential use in biomedical applications. The aim of this study was to investigate the effects of AuNPs from aqueous extract of H. erinaceus on neurite outgrowth of rat pheochromocytoma (PC-12) cells. Methods The formation of AuNPs was characterized by UV–visible spectrum, energy dispersive X-ray (EDX), field-emission scanning electron microscope (FESEM), transmission electron microscopy (TEM), particle size distribution, and Fourier transform-infrared spectroscopy (FTIR). Furthermore, the neurite extension study of synthesized AuNPs was evaluated by in vitro assay. Results The AuNPs exhibited maximum absorbance between 510 and 600 nm in UV–visible spectrum. FESEM and TEM images showed the existence of nanoparticles with sizes of 20–40 nm. FTIR measurements were carried out to identify the possible biomolecules responsible for capping and efficient stabilization of the nanoparticles. The purity and the crystalline properties were confirmed by EDX diffraction analysis, which showed strong signals with energy peaks in the range of 2–2.4 keV, indicating the existence of gold atoms. The synthesized AuNPs showed significant neurite extension on PC-12 cells. Nerve growth factor 50 ng/mL was used as a positive control. Treatment with different concentrations (nanograms) of AuNPs resulted in neuronal differentiation and neuronal elongation. AuNPs induced maximum neurite outgrowth of 13% at 600 ng/mL concentration. Conclusion In this study, the AuNPs synthesis was achieved by a simple, low-cost, and rapid bioreduction approach. AuNPs were shown to have potential neuronal differentiation and

  19. Tissue-specific neuro-glia interactions determine neurite differentiation in ganglion cells.

    PubMed

    Steinbach, K; Bauch, H; Stier, H; Schlosshauer, B

    2001-03-01

    Guided formation and extension of axons versus dendrites is considered crucial for structuring the nervous system. In the chick visual system, retinal ganglion cells (RGCs) extend their axons into the tectum opticum, but not into glial somata containing retina layers. We addressed the question whether the different glia of retina and tectum opticum differentially affect axon growth. Glial cells were purified from retina and tectum opticum by complement-mediated cytolysis of non-glial cells. RGCs were purified by enzymatic delayering from flat mounted retina. RGCs were seeded onto retinal versus tectal glia monolayers. Subsequent neuritic differentiation was analysed by immunofluorescence microscopy and scanning electron microscopy. Qualitative and quantitative evaluation revealed that retinal glia somata inhibited axons. Time-lapse video recording indicated that axonal inhibition was based on the collapse of lamellipodia- and filopodia-rich growth cones of axons. In contrast to retinal glia, tectal glia supported axonal extension. Notably, retinal glia were not inhibitory for neurons in general, because in control experiments axon extension of dorsal root ganglia was not hampered. Therefore, the axon inhibition by retinal glia was neuron type-specific. In summary, the data demonstrate that homotopic (retinal) glia somata inhibit axonal outgrowth of RGCs, whereas heterotopic (tectal) glia of the synaptic target area support RGC axon extension. The data underscore the pivotal role of glia in structuring the developing nervous system. PMID:11322389

  20. Morphine Enhances HIV-1SF162-Mediated Neuron Death and Delays Recovery of Injured Neurites

    PubMed Central

    Masvekar, Ruturaj R.; El-Hage, Nazira; Hauser, Kurt F.; Knapp, Pamela E.

    2014-01-01

    HIV-1 enters the CNS soon after initial systemic infection; within the CNS parenchyma infected and/or activated perivascular macrophages, microglia and astrocytes release viral and cellular toxins that drive secondary toxicity in neurons and other cell types. Our previous work has largely modeled HIV-neuropathology using the individual viral proteins Tat or gp120, with murine striatal neurons as targets. To model disease processes more closely, the current study uses supernatant from HIV-1-infected cells. Supernatant from HIV-1SF162-infected differentiated-U937 cells (HIV+sup) was collected and p24 level was measured by ELISA to assess the infection. Injection drug abuse is a significant risk factor for HIV-infection, and opiate drug abusers show increased HIV-neuropathology, even with anti-retroviral treatments. We therefore assessed HIV+sup effects on neuronal survival and neurite growth/pruning with or without concurrent exposure to morphine, an opiate that preferentially acts through µ-opioid receptors. Effects of HIV+sup ± morphine were assessed on neuronal populations, and also by time-lapse imaging of individual cells. HIV+sup caused dose-dependent toxicity over a range of p24 levels (10–500 pg/ml). Significant interactions occurred with morphine at lower p24 levels (10 and 25 pg/ml), and GSK3β was implicated as a point of convergence. In the presence of glia, selective neurotoxic measures were significantly enhanced and interactions with morphine were also augmented, perhaps related to a decreased level of BDNF. Importantly, the arrest of neurite growth that occurred with exposure to HIV+sup was reversible unless neurons were continuously exposed to morphine. Thus, while reducing HIV-infection levels may be protective, ongoing exposure to opiates may limit recovery. Opiate interactions observed in this HIV-infective environment were similar, though not entirely concordant, with Tat/gp120 interactions reported previously, suggesting unique interactions

  1. Effects of 60-GHz millimeter waves on neurite outgrowth in PC12 cells using high-content screening.

    PubMed

    Haas, Alexis J; Le Page, Yann; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves

    2016-04-01

    Technologies for wireless telecommunication systems using millimeter waves (MMW) will be widely deployed in the near future. Forthcoming applications in this band, especially around 60GHz, are mainly developed for high data-rate local and body-centric telecommunications. At those frequencies, electromagnetic radiations have a very shallow penetration into biological tissues, making skin keratinocytes, and free nerve endings of the upper dermis the main targets of MMW. Only a few studies assessed the impact of MMW on neuronal cells, and none of them investigated a possible effect on neuronal differentiation. We used a neuron-like cell line (PC12), which undergoes neuronal differentiation when treated with the neuronal growth factor (NGF). PC12 cells were exposed at 60.4GHz for 24h, at an incident power density averaged over the cell monolayer of 10mW/cm(2). Using a large scale cell-by-cell analysis based on high-content screening microscopy approach, we assessed potential effects of MMW on PC12 neurite outgrowth and cytoskeleton protein expression. No differences were found in protein expression of the neuronal marker β3-tubulin nor in internal expression control β-tubulin. On the other hand, our data showed a slight increase, although insignificant, in neurite outgrowth, induced by MMW exposure. However, experimental controls demonstrated that this increase was related to heating. PMID:26921450

  2. Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo.

    PubMed

    Peterson, Sheri L; Nguyen, Hal X; Mendez, Oscar A; Anderson, Aileen J

    2015-03-11

    Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI. PMID:25762679

  3. Complement Protein C1q Modulates Neurite Outgrowth In Vitro and Spinal Cord Axon Regeneration In Vivo

    PubMed Central

    Peterson, Sheri L.; Nguyen, Hal X.; Mendez, Oscar A.

    2015-01-01

    Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI. PMID:25762679

  4. Cocaine- and amphetamine-regulated transcript facilitates the neurite outgrowth in cortical neurons after oxygen and glucose deprivation through PTN-dependent pathway.

    PubMed

    Wang, Y; Qiu, B; Liu, J; Zhu, Wei-Guo; Zhu, S

    2014-09-26

    Cocaine- and amphetamine-regulated transcript (CART) is a neuropeptide that plays neuroprotective roles in cerebral ischemia and reperfusion (I/R) injury in animal models or oxygen and glucose deprivation (OGD) in cultured neurons. Recent data suggest that intranasal CART treatment facilitates neuroregeneration in stroke brain. However, little is known about the effects of post-treatment with CART during the neuronal recovery after OGD and reoxygenation in cultured primary cortical neurons. The present study was to investigate the role of CART treated after OGD injury in neurons. Primary mouse cortical neurons were subjected to OGD and then treated with CART. Our data show that post-treatment with CART reduced the neuronal apoptosis caused by OGD injury. In addition, CART repaired OGD-impaired cortical neurons by increasing the expression of growth-associated protein 43 (GAP43), which promotes neurite outgrowth. This effect depends on pleiotrophin (PTN) as siRNA-mediated PTN knockdown totally abolished the increase in CART-stimulated GAP43 protein levels. In summary, our findings demonstrate that CART repairs the neuronal injury after OGD by facilitating neurite outgrowth through PTN-dependent pathway. The role for CART in neurite outgrowth makes it a new potential therapeutic agent for the treatment of neurodegenerative diseases. PMID:25010400

  5. ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expression in vitro

    PubMed Central

    Jia, Xu-feng; Ye, Fei; Wang, Yan-bo; Feng, Da-xiong

    2016-01-01

    Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase (ROCK) pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway. PMID:27482229

  6. ANALYSIS OF THE STRUCTURE OF MAGNETIC FIELDS THAT INDUCED INHIBITION OF STIMULATED NEURITE OUTGROWTH

    EPA Science Inventory

    The important experiments showing nonlinear amplitude dependences of the neurite outgrowth in pheochromocytoma nerve cells due to ELF magnetic field exposure had been carried out in a nonuniform ac magnetic field. The nonuniformity entailed larger than expected variances in magne...

  7. Morphological assessment of neurite outgrowth in hippocampal neuron-astrocyte co-cultures.

    PubMed

    Giordano, Gennaro; Costa, Lucio G

    2012-05-01

    Neurite outgrowth is a fundamental event in brain development, as well as in regeneration of damaged neurons. Astrocytes play a major role in neuritogenesis, by expressing and releasing factors that facilitate neurite outgrowth, such as extracellular matrix proteins, and factors that can inhibit neuritogenesis, such as the chondroitin sulfate proteoglycan neurocan. In this unit we describe a noncontact co-culture system of hippocampal neurons and cortical (or hippocampal) astrocytes for measurement of neurite outgrowth. Hippocampal pyramidal neurons are plated on glass coverslips, which are inverted onto an astrocyte feeder layer, allowing exposure of neurons to astrocyte-derived factors without direct contact between these two cell types. After co-culture, neurons are stained and photographed, and processes are assessed morphologically using Metamorph software. This method allows exposing astrocytes to various agents before co-culture in order to assess how these exposures may influence the ability of astrocytes to foster neurite outgrowth. PMID:22549268

  8. Neurotrophic effect of gonadotropin-releasing hormone on neurite extension and neuronal migration of embryonic gonadotropin-releasing hormone neurons in chick olfactory nerve bundle culture.

    PubMed

    Kanaho, Yoh-Ichiro; Enomoto, Masahiro; Endo, Daisuke; Maehiro, Sayaka; Park, Min Kyun; Murakami, Shizuko

    2009-08-01

    Hypothalamic gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in regulating the reproductive function of vertebrates. These neurons are known to originate in the olfactory placode and migrate along olfactory-related axons to reach the forebrain during embryonic development. Although GnRH is suggested to be secreted during such migration, its physiological significance is unknown. This point is difficult to explore in vivo because recent studies suggest that GnRH is an important factor for normal brain development and that modification of the embryonic GnRH system by exogenous GnRH analogue or genetic methods would result in dysgenesis of the brain. Therefore, to study the role of GnRH in the migratory process of GnRH neurons, we established an in vitro chick embryonic olfactory nerve bundle explant model. Embryonic day 7.5-8 olfactory nerve bundles were cultured in a mixture of Matrigel and collagen gel. At day 3 of culture, GnRH neurons extended their unbranched neurites and migrated out from both edges of the explant. The nature of neurite extension and migratory behavior of GnRH neurons was well maintained in the gel containing 25% Matrigel and 50% collagen. With this culture system, we examined the effect of GnRH on the migrating GnRH neurons. Cetrorelix, a GnRH antagonist, was found to inhibit significantly neurite growth and neuronal migration of GnRH neurons, the effects of which were repressed by the addition of chicken GnRH-I. These results suggest that GnRH functions as one of the regulating factors of GnRH neuronal development by promoting neurite extension and neuronal migration. PMID:19301422

  9. Lipoprotein-associated lysolipids are differentially involved in high-density lipoprotein- and its oxidized form-induced neurite remodeling in PC12 cells.

    PubMed

    Sato, Koichi; Tobo, Masayuki; Mogi, Chihiro; Murata, Naoya; Kotake, Mie; Kuwabara, Atsushi; Im, Dong-Soon; Okajima, Fumikazu

    2014-03-01

    Oxidatively damaged proteins and lipid peroxidation products have been shown to accumulate in the brain of neurodegenerative diseases, such as Alzheimer's disease and multiple sclerosis, and oxidized lipoprotein is considered to be toxic and neurodegenerative. However, the role of lipoprotein and its oxidized form in neurite remodeling has not been well understood. In the present study, we have aimed to clarify whether and, if so, how high-density lipoprotein (HDL) and oxidized HDL (oxHDL) affect neuritogenesis. In the presence of nerve growth factor, exposure of PC12 cells to either HDL or oxHDL induces a rapid neurite retraction, which is followed by re-outgrowth of neurites in either case; however, oxHDL-treated cells exhibit much longer outgrowths than do basal and HDL-treated cells. Thus, processes in the morphological changes of neuronal cells after lipoprotein treatment are composed of two phases: the reversible retraction phase and the extension phase. Characterization of the active fractions of lipids and experiments with desensitization and knockdown of receptors have indicated that the reversible retraction phase involves mainly sphingosine 1-phosphate for HDL and lysophosphatidic acid for oxHDL. The change in the components responsible for the retraction response is comparable with the change in sphingosine 1-phosphate and lysophosphatidic acid contents by the oxidation of HDL. In the extension phase, lysophosphatidylcholine, which is increased by the oxidation of HDL, may play a stimulatory role in neurite outgrowth. We conclude that lipoprotein and its oxidized form differentially regulate neuritogenesis through lipoprotein-associated lysolipid molecules. PMID:24589770

  10. Hypothermia-induced neurite outgrowth is mediated by tumor necrosis factor-alpha.

    PubMed

    Schmitt, Katharina R L; Boato, Francesco; Diestel, Antje; Hechler, Daniel; Kruglov, Andrei; Berger, Felix; Hendrix, Sven

    2010-07-01

    Systemic or brain-selective hypothermia is a well-established method for neuroprotection after brain trauma. There is increasing evidence that hypothermia exerts beneficial effects on the brain and may also support regenerative responses after brain damage. Here, we have investigated whether hypothermia influences neurite outgrowth in vitro via modulation of the post-injury cytokine milieu. Organotypic brain slices were incubated: deep hypothermia (2 h at 17 degrees C), rewarming (2 h up to 37 degrees C), normothermia (20 h at 37 degrees C). Neurite density and cytokine release (IL 1beta, IL-6, IL-10, and TNF-alpha) were investigated after 24 h. For functional analysis mice deficient in NT-3/NT-4 and TNF-alpha as well as the TNF-alpha inhibitor etanercept were used. Hypothermia led to a significant increase of neurite outgrowth, which was independent of neurotrophin signaling. In contrast to other cytokines investigated, TNF-alpha secretion by organotypic brain slices was significantly increased after deep hypothermia. Moreover, hypothermia-induced neurite extension was abolished after administration of the TNF-alpha inhibitor and in TNF-alpha knockout mice. We demonstrate that TNF-alpha is responsible for inducing neurite outgrowth in the context of deep hypothermia and rewarming. These data suggest that hypothermia not only exerts protective effects in the CNS but may also support neurite outgrowth as a potential mechanism of regeneration. PMID:20070303

  11. Self-aligned Schwann cell monolayers demonstrate an inherent ability to direct neurite outgrowth

    NASA Astrophysics Data System (ADS)

    Seggio, A. M.; Narayanaswamy, A.; Roysam, B.; Thompson, D. M.

    2010-08-01

    In vivo nerve guidance channel studies have identified Schwann cell (SC) presence as an integral factor in axonal number and extension in an injury site, and in vitro studies have provided evidence that oriented SCs can direct neurite outgrowth. However, traditional methods used to create oriented SC monolayers (e.g. micropatterns/microtopography) potentially introduce secondary guidance cues to the neurons that are difficult to de-couple. Although SCs expanded on uniform laminin-coated coverslips lack a global orientation, the monolayers contain naturally formed regions of locally oriented cells that can be used to investigate SC-mediated neurite guidance. In this work, novel image analysis techniques have been developed to quantitatively assess local neurite orientation with respect to the underlying regional orientation of the Schwann cell monolayer. Results confirm that, in the absence of any secondary guidance cues, a positive correlation exists between neurite outgrowth and regional orientation of the SC monolayer. Thus, SCs alone possess an inherent ability to direct neurite outgrowth, and expansion of the co-culture-based quantitative method described can be used to further deconstruct specific biomolecular mechanisms of neurite guidance.

  12. MorphoNeuroNet: an automated method for dense neurite network analysis.

    PubMed

    Pani, Giuseppe; De Vos, Winnok H; Samari, Nada; de Saint-Georges, Louis; Baatout, Sarah; Van Oostveldt, Patrick; Benotmane, Mohammed Abderrafi

    2014-02-01

    High content cell-based screens are rapidly gaining popularity in the context of neuronal regeneration studies. To analyze neuronal morphology, automatic image analysis pipelines have been conceived, which accurately quantify the shape changes of neurons in cell cultures with non-dense neurite networks. However, most existing methods show poor performance for well-connected and differentiated neuronal networks, which may serve as valuable models for inter alia synaptogenesis. Here, we present a fully automated method for quantifying the morphology of neurons and the density of neurite networks, in dense neuronal cultures, which are grown for more than 10 days. MorphoNeuroNet, written as a script for ImageJ, Java based freeware, automatically determines various morphological parameters of the soma and the neurites (size, shape, starting points, and fractional occupation). The image analysis pipeline consists of a multi-tier approach in which the somas are segmented by adaptive region growing using nuclei as seeds, and the neurites are delineated by a combination of various intensity and edge detection algorithms. Quantitative comparison showed a superior performance of MorphoNeuroNet to existing analysis tools, especially for revealing subtle changes in thin neurites, which have weak fluorescence intensity compared to the rest of the network. The proposed method will help determining the effects of compounds on cultures with dense neurite networks, thereby boosting physiological relevance of cell-based assays in the context of neuronal diseases. PMID:24222510

  13. Multiscale Analysis of Neurite Orientation and Spatial Organization in Neuronal Images.

    PubMed

    Singh, Pankaj; Negi, Pooran; Laezza, Fernanda; Papadakis, Manos; Labate, Demetrio

    2016-10-01

    The spatial organization of neurites, the thin processes (i.e., dendrites and axons) that stem from a neuron's soma, conveys structural information required for proper brain function. The alignment, direction and overall geometry of neurites in the brain are subject to continuous remodeling in response to healthy and noxious stimuli. In the developing brain, during neurogenesis or in neuroregeneration, these structural changes are indicators of the ability of neurons to establish axon-to-dendrite connections that can ultimately develop into functional synapses. Enabling a proper quantification of this structural remodeling would facilitate the identification of new phenotypic criteria to classify developmental stages and further our understanding of brain function. However, adequate algorithms to accurately and reliably quantify neurite orientation and alignment are still lacking. To fill this gap, we introduce a novel algorithm that relies on multiscale directional filters designed to measure local neurites orientation over multiple scales. This innovative approach allows us to discriminate the physical orientation of neurites from finer scale phenomena associated with local irregularities and noise. Building on this multiscale framework, we also introduce a notion of alignment score that we apply to quantify the degree of spatial organization of neurites in tissue and cultured neurons. Numerical codes were implemented in Python and released open source and freely available to the scientific community. PMID:27369547

  14. Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

    PubMed Central

    Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide

    2013-01-01

    Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca2+ signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB. PMID:24116072

  15. Semiconductor nanomembrane tubes: three-dimensional confinement for controlled neurite outgrowth.

    PubMed

    Yu, Minrui; Huang, Yu; Ballweg, Jason; Shin, Hyuncheol; Huang, Minghuang; Savage, Donald E; Lagally, Max G; Dent, Erik W; Blick, Robert H; Williams, Justin C

    2011-04-26

    In many neural culture studies, neurite migration on a flat, open surface does not reflect the three-dimensional (3D) microenvironment in vivo. With that in mind, we fabricated arrays of semiconductor tubes using strained silicon (Si) and germanium (Ge) nanomembranes and employed them as a cell culture substrate for primary cortical neurons. Our experiments show that the SiGe substrate and the tube fabrication process are biologically viable for neuron cells. We also observe that neurons are attracted by the tube topography, even in the absence of adhesion factors, and can be guided to pass through the tubes during outgrowth. Coupled with selective seeding of individual neurons close to the tube opening, growth within a tube can be limited to a single axon. Furthermore, the tube feature resembles the natural myelin, both physically and electrically, and it is possible to control the tube diameter to be close to that of an axon, providing a confined 3D contact with the axon membrane and potentially insulating it from the extracellular solution. PMID:21366271

  16. Salubrinal inhibits the expression of proteoglycans and favors neurite outgrowth from cortical neurons in vitro.

    PubMed

    Barreda-Manso, M Asunción; Yanguas-Casás, Natalia; Nieto-Sampedro, Manuel; Romero-Ramírez, Lorenzo

    2015-07-01

    After CNS injury, astrocytes and mesenchymal cells attempt to restore the disrupted glia limitans by secreting proteoglycans and extracellular matrix proteins (ECMs), forming the so-called glial scar. Although the glial scar is important in sealing the lesion, it is also a physical and functional barrier that prevents axonal regeneration. The synthesis of secretory proteins in the RER is under the control of the initiation factor of translation eIF2α. Inhibiting the synthesis of secretory proteins by increasing the phosphorylation of eIF2α, might be a pharmacologically efficient way of reducing proteoglycans and other profibrotic proteins present in the glial scar. Salubrinal, a neuroprotective drug, decreased the expression and secretion of proteoglycans and other profibrotic proteins induced by EGF or TGFβ, maintaining eIF2α phosphorylated. Besides, Salubrinal also reduced the transcription of proteoglycans and other profibrotic proteins, suggesting that it induced the degradation of non-translated mRNA. In a model in vitro of the glial scar, cortical neurons grown on cocultures of astrocytes and fibroblasts with TGFβ treated with Salubrinal, showed increased neurite outgrowth compared to untreated cells. Our results suggest that Salubrinal may be considered of therapeutic value facilitating axonal regeneration, by reducing overproduction and secretion of proteoglycans and profibrotic protein inhibitors of axonal growth. PMID:25882497

  17. Surface Microstructures on Planar Substrates and Textile Fibers Guide Neurite Outgrowth: A Scaffold Solution to Push Limits of Critical Nerve Defect Regeneration?

    PubMed Central

    Weigel, Stefan; Tobler, Ursina; Yao, Li; Wiesli, Manuel; Lehnert, Thomas; Pandit, Abhay; Bruinink, Arie

    2012-01-01

    The treatment of critical size peripheral nerve defects represents one of the most serious problems in neurosurgery. If the gap size exceeds a certain limit, healing can't be achieved. Connection mismatching may further reduce the clinical success. The present study investigates how far specific surface structures support neurite outgrowth and by that may represent one possibility to push distance limits that can be bridged. For this purpose, growth cone displacement of fluorescent embryonic chicken spinal cord neurons was monitored using time-lapse video. In a first series of experiments, parallel patterns of polyimide ridges of different geometry were created on planar silicon oxide surfaces. These channel-like structures were evaluated with and without amorphous hydrogenated carbon (a-C:H) coating. In a next step, structured and unstructured textile fibers were investigated. All planar surface materials (polyimide, silicon oxide and a-C:H) proved to be biocompatible, i.e. had no adverse effect on nerve cultures and supported neurite outgrowth. Mean growth cone migration velocity measured on 5 minute base was marginally affected by surface structuring. However, surface structure variability, i.e. ridge height, width and inter-ridge spacing, significantly enhanced the resulting net velocity by guiding the growth cone movement. Ridge height and inter-ridge distance affected the frequency of neurites crossing over ridges. Of the evaluated dimensions ridge height, width, and inter-ridge distance of respectively 3, 10, and 10 µm maximally supported net axon growth. Comparable artificial grooves, fabricated onto the surface of PET fibers by using an excimer laser, showed similar positive effects. Our data may help to further optimize surface characteristics of artificial nerve conduits and bioelectronic interfaces. PMID:23251379

  18. Surface microstructures on planar substrates and textile fibers guide neurite outgrowth: a scaffold solution to push limits of critical nerve defect regeneration?

    PubMed

    Weigel, Stefan; Osterwalder, Thomas; Tobler, Ursina; Yao, Li; Wiesli, Manuel; Lehnert, Thomas; Pandit, Abhay; Bruinink, Arie

    2012-01-01

    The treatment of critical size peripheral nerve defects represents one of the most serious problems in neurosurgery. If the gap size exceeds a certain limit, healing can't be achieved. Connection mismatching may further reduce the clinical success. The present study investigates how far specific surface structures support neurite outgrowth and by that may represent one possibility to push distance limits that can be bridged. For this purpose, growth cone displacement of fluorescent embryonic chicken spinal cord neurons was monitored using time-lapse video. In a first series of experiments, parallel patterns of polyimide ridges of different geometry were created on planar silicon oxide surfaces. These channel-like structures were evaluated with and without amorphous hydrogenated carbon (a-C:H) coating. In a next step, structured and unstructured textile fibers were investigated. All planar surface materials (polyimide, silicon oxide and a-C:H) proved to be biocompatible, i.e. had no adverse effect on nerve cultures and supported neurite outgrowth. Mean growth cone migration velocity measured on 5 minute base was marginally affected by surface structuring. However, surface structure variability, i.e. ridge height, width and inter-ridge spacing, significantly enhanced the resulting net velocity by guiding the growth cone movement. Ridge height and inter-ridge distance affected the frequency of neurites crossing over ridges. Of the evaluated dimensions ridge height, width, and inter-ridge distance of respectively 3, 10, and 10 µm maximally supported net axon growth. Comparable artificial grooves, fabricated onto the surface of PET fibers by using an excimer laser, showed similar positive effects. Our data may help to further optimize surface characteristics of artificial nerve conduits and bioelectronic interfaces. PMID:23251379

  19. Specificity of prenatal cocaine on inhibition of locus coeruleus neurite outgrowth.

    PubMed

    Dey, S; Mactutus, C F; Booze, R M; Snow, D M

    2006-01-01

    Prenatal cocaine exposure induces alterations in attentional function that presumably involve locus coeruleus noradrenergic neurons and their projections. Previous reports indicate that embryonic rat locus coeruleus neurons exposed to cocaine, both in vitro and in vivo, showed in decreased cell survival and inhibition of neurite outgrowth, and that the effects were most deleterious during early gestation. The present study performed in vitro addressed the specificity of the inhibitory effects of cocaine by comparing locus coeruleus neurite formation and extension to that of dopaminergic substantia nigra neurons following exposure to a physiologically-relevant dose of cocaine (500 ng/ml, two times a day, for four days) during peak neuritogenesis. Following cocaine treatment, immunocytochemistry (anti-norepinephrine antibody to locus coeruleus; anti-tyrosine hydroxylase antibody to substantia nigra) and image analysis were performed to measure a variety of neurite outgrowth parameters. For locus coeruleus neurons, cocaine treatment decreased the 1) number of cells initiating neurites [P<0.001], 2) mean number [P<0.05] and length of neurites [P<0.0001], 3) mean number [P<0.0016] and length of branched neurites [P<0.0006], and 4) mean length of the longest neurites [P<0.0001]. In comparison, substantia nigra neurons were not significantly affected by cocaine for any of the parameters examined. More importantly, a significant interaction between cocaine treatment and brain region was observed [P<0.0002] indicating greater vulnerability of locus coeruleus, relative to substantia nigra neurons, to cocaine exposure. These data support our hypothesis that cocaine targets the noradrenergic system by negatively regulating locus coeruleus neuronal outgrowth, which likely affects pathfinding, synaptic connectivity, and ultimately attentional behavior in cocaine-exposed offspring. PMID:16483722

  20. Gene dosage-dependent rescue of HSP neurite defects in SPG4 patients’ neurons

    PubMed Central

    Havlicek, Steven; Kohl, Zacharias; Mishra, Himanshu K.; Prots, Iryna; Eberhardt, Esther; Denguir, Naime; Wend, Holger; Plötz, Sonja; Boyer, Leah; Marchetto, Maria C.N.; Aigner, Stefan; Sticht, Heinrich; Groemer, Teja W.; Hehr, Ute; Lampert, Angelika; Schlötzer-Schrehardt, Ursula; Winkler, Jürgen; Gage, Fred H.; Winner, Beate

    2014-01-01

    The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients. PMID:24381312

  1. Pavarotti/MKLP1 regulates microtubule sliding and neurite outgrowth in Drosophila neurons

    PubMed Central

    del Castillo, Urko; Lu, Wen; Winding, Michael; Lakonishok, Margot; Gelfand, Vladimir I.

    2014-01-01

    Summary Recently, we demonstrated that kinesin-1 can slide microtubules against each other providing the mechanical force required for initial neurite extension in Drosophila neurons. This sliding is only observed in young neurons actively forming neurites and is dramatically downregulated in older neurons. The downregulation is not caused by the global shut-down of kinesin-1, as the ability of kinesin-1 to transport membrane organelles is not diminished in mature neurons, suggesting that microtubule sliding is regulated by a dedicated mechanism [1]. Here, we have identified the “mitotic” kinesin Pavarotti (Pav-KLP) as an inhibitor of kinesin-1-driven microtubule sliding. Depletion of Pav-KLP in neurons strongly stimulated the sliding of long microtubules and neurite outgrowth, while its ectopic overexpression in the cytoplasm blocked both of these processes. Furthermore, postmitotic depletion of Pav-KLP in Drosophila neurons in vivo reduced embryonic/larval viability, with only a few animals surviving to the third instar larval stage. A detailed examination of motor neurons in the surviving larvae revealed the overextension of axons and mistargeting of neuromuscular junctions, resulting in uncoordinated locomotion. Taken together, our results identify a new role for Pav-KLP as a negative regulator of kinesin-1 driven neurite formation. These data suggest an important parallel between long microtubule-microtubule sliding in anaphase B and sliding of interphase microtubules during neurite formation. PMID:25557664

  2. IL-1{beta} promotes neurite outgrowth by deactivating RhoA via p38 MAPK pathway

    SciTech Connect

    Temporin, Ko; Tanaka, Hiroyuki Kuroda, Yusuke; Okada, Kiyoshi; Yachi, Koji; Moritomo, Hisao; Murase, Tsuyoshi; Yoshikawa, Hideki

    2008-01-11

    Expression of the pro-inflammatory cytokine interleukin-1 beta (IL-1{beta}) is increased following the nervous system injury. Generally IL-1{beta} induces inflammation, leading to neural degeneration, while several neuropoietic effects have also been reported. Although neurite outgrowth is an important step in nerve regeneration, whether IL-1{beta} takes advantages on it is unclear. Now we examine how it affects neurite outgrowth. Following sciatic nerve injury, expression of IL-1{beta} is increased in Schwann cells around the site of injury, peaking 1 day after injury. In dorsal root ganglion (DRG) neurons and cerebellar granule neurons (CGNs), neurite outgrowth is inhibited by the addition of myelin-associated glycoprotein (MAG), activating RhoA. IL-1{beta} overcomes MAG-induced neurite outgrowth inhibition, by deactivating RhoA. Intracellular signaling experiments reveal that p38 MAPK, and not nuclear factor-kappa B (NF-{kappa}B), mediated this effect. These findings suggest that IL-1{beta} may contribute to nerve regeneration by promoting neurite outgrowth following nerve injury.

  3. Berberine regulates neurite outgrowth through AMPK-dependent pathways by lowering energy status

    SciTech Connect

    Lu, Jiaqi; Cao, Yuanzhao; Cheng, Kuoyuan; Xu, Bo; Wang, Tianchang; Yang, Qi; Yang, Qin; Feng, Xudong; Xia, Qing

    2015-06-10

    As a widely used anti-bacterial agent and a metabolic inhibitor as well as AMP-activated protein kinase (AMPK) activator, berberine (BBR) has been shown to cross the blood–brain barrier. Its efficacy has been investigated in various disease models of the central nervous system. Neurite outgrowth is critical for nervous system development and is a highly energy-dependent process regulated by AMPK-related pathways. In the present study, we aimed to investigate the effects of BBR on AMPK activation and neurite outgrowth in neurons. The neurite outgrowth of primary rat cortical neurons at different stages of polarization was monitored after exposure of BBR. Intracellular energy level, AMPK activation and polarity-related pathways were also inspected. The results showed that BBR suppressed neurite outgrowth and affected cytoskeleton stability in the early stages of neuronal polarization, which was mediated by lowered energy status and AMPK activation. Liver kinase B1 and PI3K–Akt–GSK3β signaling pathways were also involved. In addition, mitochondrial dysfunction and endoplasmic reticulum stress contributed to the lowered energy status induced by BBR. This study highlighted the knowledge of the complex activities of BBR in neurons and corroborated the significance of energy status during the neuronal polarization. - Highlights: • BBR inhibited neurite outgrowth in early stages of neuronal development. • Lowered neuronal energy status was induced by BBR treatment. • Neuronal energy stress induced by BBR activated AMPK-related pathways. • BBR induced mitochondrial dysfunction and endoplasmic reticulum stress.

  4. Mutations changing tropomodulin affinity for tropomyosin alter neurite formation and extension.

    PubMed

    Moroz, Natalia; Guillaud, Laurent; Desai, Brinda; Kostyukova, Alla S

    2013-01-01

    Assembly of the actin cytoskeleton is an important part of formation of neurites in developing neurons. Tropomodulin, a tropomyosin-dependent capping protein for the pointed end of the actin filament, is one of the key players in this process. Tropomodulin binds tropomyosin in two binding sites. Tmod1 and Tmod2, tropomodulin isoforms found in neurons, were overexpressed in PC12 cells, a model system for neuronal differentiation. Tmod1 did not affect neuronal differentiation; while cells expressing Tmod2 showed a significant reduction in the number and the length of neurites. Both tropomodulins bind short α-, γ- and δ-tropomyosin isoforms. Mutations in one of the tropomyosin-binding sites of Tmod1, which increased its affinity to short γ- and δ-tropomyosin isoforms, caused a decrease in binding short α-tropomyosin isoforms along with a 2-fold decrease in the length of neurites. Our data demonstrate that Tmod1 is involved in neuronal differentiation for proper neurite formation and outgrowth, and that Tmod2 inhibits these processes. The mutations in the tropomyosin-binding site of Tmod1 impair neurite outgrowth, suggesting that the integrity of this binding site is critical for the proper function of Tmod1 during neuronal differentiation. PMID:23638401

  5. Bingham-NODDI: Mapping anisotropic orientation dispersion of neurites using diffusion MRI.

    PubMed

    Tariq, Maira; Schneider, Torben; Alexander, Daniel C; Gandini Wheeler-Kingshott, Claudia A; Zhang, Hui

    2016-06-01

    This paper presents Bingham-NODDI, a clinically-feasible technique for estimating the anisotropic orientation dispersion of neurites. Direct quantification of neurite morphology on clinical scanners was recently realised by a diffusion MRI technique known as neurite orientation dispersion and density imaging (NODDI). However in its current form NODDI cannot estimate anisotropic orientation dispersion, which is widespread in the brain due to common fanning and bending of neurites. This work proposes Bingham-NODDI that extends the NODDI formalism to address this limitation. Bingham-NODDI characterises anisotropic orientation dispersion by utilising the Bingham distribution to model neurite orientation distribution. The new model estimates the extent of dispersion about the dominant orientation, separately along the primary and secondary dispersion orientations. These estimates are subsequently used to estimate the overall dispersion about the dominant orientation and the dispersion anisotropy. We systematically evaluate the ability of the new model to recover these key parameters of anisotropic orientation dispersion with standard NODDI protocol, both in silico and in vivo. The results demonstrate that the parameters of the proposed model can be estimated without additional acquisition requirements over the standard NODDI protocol. Thus anisotropic dispersion can be determined and has the potential to be used as a marker for normal brain development and ageing or in pathology. We additionally find that the original NODDI model is robust to the effects of anisotropic orientation dispersion, when the quantification of anisotropic dispersion is not of interest. PMID:26826512

  6. Tiam1 mediates neurite outgrowth induced by ephrin-B1 and EphA2

    PubMed Central

    Tanaka, Masamitsu; Ohashi, Riuko; Nakamura, Ritsuko; Shinmura, Kazuya; Kamo, Takaharu; Sakai, Ryuichi; Sugimura, Haruhiko

    2004-01-01

    Bidirectional signals mediated by Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, play pivotal roles in the formation of neural networks by induction of both collapse and elongation of neurites. However, the downstream molecular modules to deliver these cues are largely unknown. We report here that the interaction of a Rac1-specific guanine nucleotide-exchanging factor, Tiam1, with ephrin-B1 and EphA2 mediates neurite outgrowth. In cells coexpressing Tiam1 and ephrin-B1, Rac1 is activated by the extracellular stimulation of clustered soluble EphB2 receptors. Similarly, soluble ephrin-A1 activates Rac1 in cells coexpressing Tiam1 and EphA2. Cortical neurons from the E14 mouse embryos and neuroblastoma cells significantly extend neurites when placed on surfaces coated with the extracellular domain of EphB2 or ephrin-A1, which were abolished by the forced expression of the dominant-negative mutant of ephrin-B1 or EphA2. Furthermore, the introduction of a dominant-negative form of Tiam1 also inhibits neurite outgrowth induced by the ephrin-B1 and EphA2 signals. These results indicate that Tiam1 is required for neurite outgrowth induced by both ephrin-B1-mediated reverse signaling and EphA2-mediated forward signaling. PMID:14988728

  7. Two stages in neurite formation distinguished by differences in tubulin metabolism.

    PubMed

    Sekimoto, S; Tashiro, T; Komiya, Y

    1995-01-01

    Changes in tubulin solubility during neurite formation were studied biochemically using rat dorsal root ganglion neurons in culture. When fractionated with Ca(2+)-containing buffer at low temperature, a considerable proportion of total cellular tubulin was recovered in the insoluble fraction. We designated this cold/Ca(2+)-insoluble tubulin (InsT) and distinguished it from cold/Ca(2+)-soluble tubulin (SoIT). From the relative amount of InsT, neurite formation was found to proceed through two distinct stages. The first 6 days after plating (stage 1) in which the proportion of InsT increased dramatically (from 5 to 60%) coincided with neurite outgrowth. In the following period (stage 2), a constant level of InsT was maintained, whereas neurite maturation took place. Pulse-labeling experiments further revealed that the two stages differed significantly in terms of tubulin metabolism. High rates of synthesis as well as conversion from SoIT to InsT were observed in stage 1, whereas stage 2 was characterized by a decrease in both of these rates and an increase in the rate of degradation. The results show for the first time the coordinated changes in tubulin metabolism that underlie the process of neurite formation. PMID:7798932

  8. Lignosus rhinocerus (Cooke) Ryvarden: A Medicinal Mushroom That Stimulates Neurite Outgrowth in PC-12 Cells

    PubMed Central

    Eik, Lee-Fang; Naidu, Murali; David, Pamela; Wong, Kah-Hui; Tan, Yee-Shin; Sabaratnam, Vikineswary

    2012-01-01

    A national treasure mushroom, Lignosus rhinocerus, has been used to treat variety of ailments by local and indigenous communities in Malaysia. The aim of this study was to investigate the potential of the most valuable part of L. rhinocerus, the sclerotium, on neurite outgrowth activity by using PC-12Adh cell line. Differentiated cells with one thin extension at least double the length of the cell diameter were scored positive. Our results showed that aqueous sclerotium L. rhinocerus extract induced neurite outgrowths of 24.4% and 42.1% at 20 μg/mL (w/v) of aqueous extract alone and a combination of 20 μg/mL (w/v) aqueous extract and 30 ng/mL (w/v) of NGF, respectively. Combination of NGF and sclerotium extract had additive effects and enhanced neurite outgrowth. Neuronal differentiation was demonstrated by indirect immunofluorescence of neurofilament protein. Aqueous sclerotium extract contained neuroactive compounds that stimulated neurite outgrowth in vitro. To our knowledge this is the first report on neurite-stimulating activities of L. rhinocerus. PMID:22203867

  9. A cortical astrocyte subpopulation inhibits axon growth in vitro and in vivo.

    PubMed

    Liu, Rui; Wang, Zhe; Gou, Lin; Xu, Hanpeng

    2015-08-01

    Astrocytes are the most heterogeneous and predominant glial cell type in the central nervous system. However, the functional significance of this heterogeneity remains to be elucidated. Following injury, damaged astrocytes inhibit axonal regeneration in vivo and in vitro. Cultured primary astrocytes are commonly considered good supportive substrates for neuron attachment and axon regeneration. However, it is not known whether different populations of cells in the heterogeneous astrocyte culture affect neuron behavior in the same way. In the present study, the effect of astrocyte heterogeneity on neuronal attachment and neurite outgrowth was examined using an in vitro and in vivo coculture system. In vitro, neonatal cortical astrocytes were co-cultured with purified dorsal root ganglia (DRG) neurons and astrocyte growth morphology, neuron attachment and neurite growth were evaluated. The results demonstrated that the heterogeneous astrocyte cells showed two different types of growth pattern, typical and atypical. Typical astrocytes were supportive to neuron attachment and neurite growth, which was consistent with previous studies, whereas atypical astrocytes inhibited neuron attachment and neurite growth. These inhibitory astrocytes exhibited a special growth pattern with various shapes and sizes, a high cell density, few oligodendrocytes on the top layer and occupied a smaller growth area compared with typical astrocytes. Neurites extended freely on typical supportive astrocyte populations, however, moved away when they reached atypical astrocyte growth pattern. Neurons growing on the atypical astrocyte pattern demonstrated minimal neurite outgrowth and these neurites had a dystrophic appearance, however, neuronal survival was unaffected. Immunocytochemistry studies demonstrated that these atypical inhibitory astrocytes were glial fibrillary acidic protein (GFAP) positive cells. The existence of inhibitory astrocyte subpopulations in normal astrocytes reflects the

  10. The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells

    SciTech Connect

    Marzinke, Mark A.; Clagett-Dame, Margaret

    2012-01-01

    The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21{sup Cip1}, a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G{sub 1}/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer Calmin is a retinoic acid-responsive gene. Black-Right-Pointing-Pointer Calmin promotes cell cycle exit in N2A cells. Black-Right-Pointing-Pointer Calmin overexpression increases p21Cip1 and decreases cyclin D1. Black-Right-Pointing-Pointer Calmin is required for RA-induced growth inhibition and neurite outgrowth.

  11. Bioassay, isolation and studies on the mechanism of action of neurite extension factor

    NASA Technical Reports Server (NTRS)

    Kligman, D.

    1984-01-01

    The identification and purification of molecules active in promoting neurite outgrowth requires a sensitive reproducible bioassay. A quantitative bioassay was utilized to purify a neurite extension factor (NEF) based on counting the number of phase bright neurons with processes at least equal to one cell body diameter after 20 hrs. in culture is defined, serum free medium. Using a combination of heat treatment DEAE cellulose chromatography and gel filtration, an acidic protein of M sub r = 75,000 was highly purified. Upon reduction, it yields subunits of M sub r = 37,000. Purified fractions are active half maximally at 100 ng/ml in inducing neurite outgrowth in this bioassay. Currently, monoclonal antibodies to NEF are being produced. Female Balb C mice were immunized with the antigen and fusions with mouse myeloma cells will be performed to yield hybridoma cells.

  12. Secretory phospholipases A2 induce neurite outgrowth in PC12 cells.

    PubMed Central

    Nakashima, Satoru; Ikeno, Yutaka; Yokoyama, Tatsuya; Kuwana, Masakazu; Bolchi, Angelo; Ottonello, Simone; Kitamoto, Katsuhiko; Arioka, Manabu

    2003-01-01

    sPLA(2)s (secretory phospholipases A(2)) belong to a broad and structurally diverse family of enzymes that hydrolyse the sn -2 ester bond of glycerophospholipids. We previously showed that a secreted fungal 15 kDa protein, named p15, as well as its orthologue from Streptomyces coelicolor (named Scp15) induce neurite outgrowth in PC12 cells at nanomolar concentrations. We report here that both p15 and Scp15 are members of a newly identified group of fungal/bacterial sPLA(2)s. The phospholipid-hydrolysing activity of p15 is absolutely required for neurite outgrowth induction. Mutants with a reduced PLA(2) activity exhibited a comparable reduction in neurite-inducing activity, and the ability to induce neurites closely matched the capacity of various p15 forms to promote fatty acid release from live PC12 cells. A structurally divergent member of the sPLA(2) family, bee venom sPLA(2), also induced neurites in a phospholipase activity-dependent manner, and the same effect was elicited by mouse group V and X sPLA(2)s, but not by group IB and IIA sPLA(2)s. Lysophosphatidylcholine, but not other lysophospholipids, nor arachidonic acid, elicited neurite outgrowth in an L-type Ca(2+) channel activity-dependent manner. In addition, p15-induced neuritogenesis was unaffected by various inhibitors that block arachidonic acid conversion into bioactive eicosanoids. Altogether, these results delineate a novel, Ca(2+)- and lysophosphatidylcholine-dependent neurotrophin-like role of sPLA(2)s in the nervous system. PMID:12967323

  13. Diazinon and diazoxon impair the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons

    SciTech Connect

    Pizzurro, Daniella M.; Dao, Khoi; Costa, Lucio G.

    2014-02-01

    Evidence from in vivo and epidemiological studies suggests that organophosphorus insecticides (OPs) are developmental neurotoxicants, but possible underlying mechanisms are still unclear. Astrocytes are increasingly recognized for their active role in normal neuronal development. This study sought to investigate whether the widely-used OP diazinon (DZ), and its oxygen metabolite diazoxon (DZO), would affect glial–neuronal interactions as a potential mechanism of developmental neurotoxicity. Specifically, we investigated the effects of DZ and DZO on the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons. The results show that both DZ and DZO adversely affect astrocyte function, resulting in inhibited neurite outgrowth in hippocampal neurons. This effect appears to be mediated by oxidative stress, as indicated by OP-induced increased reactive oxygen species production in astrocytes and prevention of neurite outgrowth inhibition by antioxidants. The concentrations of OPs were devoid of cytotoxicity, and cause limited acetylcholinesterase inhibition in astrocytes (18 and 25% for DZ and DZO, respectively). Among astrocytic neuritogenic factors, the most important one is the extracellular matrix protein fibronectin. DZ and DZO decreased levels of fibronectin in astrocytes, and this effect was also attenuated by antioxidants. Underscoring the importance of fibronectin in this context, adding exogenous fibronectin to the co-culture system successfully prevented inhibition of neurite outgrowth caused by DZ and DZO. These results indicate that DZ and DZO increase oxidative stress in astrocytes, and this in turn modulates astrocytic fibronectin, leading to impaired neurite outgrowth in hippocampal neurons. - Highlights: • DZ and DZO inhibit astrocyte-mediated neurite outgrowth in rat hippocampal neurons. • Oxidative stress is involved in inhibition of neuritogenesis by DZ and DZO. • DZ and DZO decrease expression of the neuritogenic

  14. ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

    SciTech Connect

    Han, Jeong-Ho; Yu, Tae-Hoon; Ryu, Hyun-Hee; Jun, Mi-Hee; Ban, Byung-Kwan; Jang, Deok-Jin; Lee, Jin-A

    2013-08-01

    Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.

  15. Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

    PubMed Central

    Shahmoradian, Sarah H.; Galiano, Mauricio R.; Wu, Chengbiao; Chen, Shurui; Rasband, Matthew N.; Mobley, William C.; Chiu, Wah

    2014-01-01

    Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts. Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders. PMID:24561719

  16. Neuritic Plaques and Cerebrovascular Amyloid in Alzheimer Disease are Antigenically Related

    NASA Astrophysics Data System (ADS)

    Wong, Caine W.; Quaranta, Vito; Glenner, George G.

    1985-12-01

    A synthetic peptide (Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr), homologous to the amino terminus of a protein purified from cerebrovascular amyloid (β protein), induced antibodies in BALB/c mice that were used immunohistochemically to stain not only amyloid-laden cerebral vessels but neuritic plaques as well. These findings suggest that the amyloid in neuritic plaques shares antigenic determinants with β protein of cerebral vessels. Since the amino acid compositions of plaque amyloid and cerebrovascular amyloid are similar, it is likely that plaque amyloid also consists of β protein. This possibility suggests a model for the pathogenesis of Alzheimer disease involving β protein.

  17. Macrophage-secreted factors induce adipocyte inflammation and insulin resistance

    SciTech Connect

    Permana, Paska A. . E-mail: Paska.Permana@med.va.gov; Menge, Christopher; Reaven, Peter D.

    2006-03-10

    Macrophage infiltration into adipose tissue increases with obesity, a condition associated with low-grade inflammation and insulin resistance. We investigated the direct effects of macrophage-secreted factors on adipocyte inflammation and insulin resistance. 3T3-L1 adipocytes incubated with media conditioned by RAW264.7 macrophages (RAW-CM) showed dramatically increased transcription of several inflammation-related genes, greater nuclear factor kappa B (NF-{kappa}B) activity, and enhanced binding of U937 monocytes. All of these effects were prevented by co-incubation with pyrrolidinedithiocarbamate, an NF-{kappa}B inhibitor. Adipocytes incubated with RAW-CM also released more non-esterified fatty acids and this increased lipolysis was not suppressed by insulin. In addition, RAW-CM treatment decreased insulin-stimulated glucose uptake in adipocytes. Taken together, these results indicate that macrophage-secreted factors induce inflammatory responses and reduce insulin responsiveness in adipocytes. These effects of macrophage-secreted factors on adipocytes may contribute significantly to the systemic inflammation and insulin resistance associated with obesity.

  18. Effects of sub-lethal neurite outgrowth inhibitory concentrations of chlorpyrifos oxon on cytoskeletal proteins and acetylcholinesterase in differentiating N2a cells

    SciTech Connect

    Flaskos, J.; Nikolaidis, E.; Harris, W.; Sachana, M.; Hargreaves, A.J.

    2011-11-15

    Previous work in our laboratory has shown that sub-lethal concentrations (1-10 {mu}M) of chlorpyrifos (CPF), diazinon (DZ) and diazinon oxon (DZO) inhibit the outgrowth of axon-like neurites in differentiating mouse N2a neuroblastoma cells concomitant with altered levels and/or phosphorylation state of axonal cytoskeleton and growth-associated proteins. The aim of the present work was to determine whether chlorpyrifos oxon (CPO) was capable of inhibiting N2a cell differentiation in a similar manner. Using experimental conditions similar to our previous work, sub-lethal concentrations (1-10 {mu}M) of CPO were found to inhibit N2a cell differentiation. However, unlike previous studies with DZ and DZO, there was a high level of sustained inhibition of acetylcholinesterase (AChE) in CPO treated cells. Impairment of neurite outgrowth was also associated with reduced levels of growth associated protein-43 and neurofilament heavy chain (NFH), and the distribution of NFH in cells stained by indirect immunofluorescence was disrupted. However, in contrast to previous findings for DZO, the absolute level of phosphorylated NFH was unaffected by CPO exposure. Taken together, the findings suggest that sub-lethal concentrations of CPO inhibit axon outgrowth in differentiating N2a cells and that this effect involves reduced levels of two proteins that play key roles in axon outgrowth and maintenance. Although the inhibition of neurite outgrowth is unlikely to involve AChE inhibition directly, further work will help to determine whether the persistent inhibition of AChE by CPO can account for the different effects induced by CPO and DZO on the levels of total and phosphorylated NFH. -- Highlights: Black-Right-Pointing-Pointer Sub-lethal levels of chlorpyrifos oxon inhibit neurite outgrowth in N2a cells Black-Right-Pointing-Pointer Acetylcholinesterase exhibits sustained inhibition throughout exposure Black-Right-Pointing-Pointer The levels of neurofilament heavy chain and GAP-43

  19. Thiazolidinediones inhibit the growth of PC12 cells both in vitro and in vivo

    SciTech Connect

    Kim, Sang Wan; Choi, Ok Kyung; Chang, Mee Soo; Shin, Chan Soo; Park, Kyong Soo; Kim, Seong Yeon

    2008-06-27

    Thiazolidinediones (TZDs) have recently been proposed as a therapy for PPAR{gamma}-expressing tumors. Pheochromocytoma (PHEO) is associated with high morbidity and mortality due to excess catecholamine production, and few effective drug therapies currently exist. We investigated the effects of TZDs on PHEO both in vitro and in vivo. PPAR{gamma} protein was expressed in human adrenal PHEO tissues as well as in rat PHEO cells, PC12. TZDs, including rosiglitazone (RGZ) and pioglitazone (PGZ), inhibited proliferation of PC12 cells in a dose-dependent manner and increased casapse-3 expression of PC12 cells. TZDs also reduced expression of cyclin E and cyclin-dependent kinase2. RGZ inhibited nerve growth factor-induced neurite outgrowth and reduced expression of catecholamine-synthesizing enzymes. Finally, rat PHEO growth generated by subcutaneous injection of PC12 cells was slowed in an RGZ-treated mouse. These data suggest that TZDs may be a promising therapeutic approach for medical treatment for PHEO.

  20. Patterned and functionalized nanofiber scaffolds in three-dimensional hydrogel constructs enhance neurite outgrowth and directional control

    NASA Astrophysics Data System (ADS)

    McMurtrey, Richard J.

    2014-12-01

    Objective. Neural tissue engineering holds incredible potential to restore functional capabilities to damaged neural tissue. It was hypothesized that patterned and functionalized nanofiber scaffolds could control neurite direction and enhance neurite outgrowth. Approach. A method of creating aligned electrospun nanofibers was implemented and fiber characteristics were analyzed using environmental scanning electron microscopy. Nanofibers were composed of polycaprolactone (PCL) polymer, PCL mixed with gelatin, or PCL with a laminin coating. Three-dimensional hydrogels were then integrated with embedded aligned nanofibers to support neuronal cell cultures. Microscopic images were captured at high-resolution in single and multi-focal planes with eGFP-expressing neuronal SH-SY5Y cells in a fluorescent channel and nanofiber scaffolding in another channel. Neuronal morphology and neurite tracking of nanofibers were then analyzed in detail. Main results. Aligned nanofibers were shown to enable significant control over the direction of neurite outgrowth in both two-dimensional (2D) and three-dimensional (3D) neuronal cultures. Laminin-functionalized nanofibers in 3D hyaluronic acid (HA) hydrogels enabled significant alignment of neurites with nanofibers, enabled significant neurite tracking of nanofibers, and significantly increased the distance over which neurites could extend. Specifically, the average length of neurites per cell in 3D HA constructs with laminin-functionalized nanofibers increased by 66% compared to the same laminin fibers on 2D laminin surfaces, increased by 59% compared to 2D laminin-coated surface without fibers, and increased by 1052% compared to HA constructs without fibers. Laminin functionalization of fibers also doubled average neurite length over plain PCL fibers in the same 3D HA constructs. In addition, neurites also demonstrated tracking directly along the fibers, with 66% of neurite lengths directly tracking laminin-coated fibers in 3D HA

  1. Guidance of dorsal root ganglion neurites and Schwann cells by isolated Schwann cell topography on poly(dimethyl siloxane) conduits and films

    NASA Astrophysics Data System (ADS)

    Richardson, J. A.; Rementer, C. W.; Bruder, Jan M.; Hoffman-Kim, D.

    2011-08-01

    Biomimetic replicas of cellular topography have been utilized to direct neurite outgrowth. Here, we cultured postnatal rat dorsal root ganglion (DRG) explants in the presence of Schwann cell (SC) topography to determine the influence of SC topography on neurite outgrowth. Four distinct poly(dimethyl siloxane) conduits were fabricated within which DRG explants were cultured. To determine the contribution of SC topographical features to neurite guidance, the extent of neurite outgrowth into unpatterned conduits, conduits with randomly oriented SC replicas, and conduits with SC replicas parallel or perpendicular to the conduit long axis was measured. Neurite directionality and outgrowth from DRG were also quantified on two-dimensional SC replicas with orientations corresponding to the four conduit conditions. Additionally, live SC migration and neurite extension from DRG on SC replicas were examined as a first step toward quantification of the interactions between live SC and navigating neurites on SC replicas. DRG neurite outgrowth and morphology within conduits and on two-dimensional SC replicas were directed by the underlying SC topographical features. Maximal neurite outgrowth and alignment to the underlying features were observed into parallel conduits and on parallel two-dimensional substrates, whereas the least extent of outgrowth was observed into perpendicular conduits and on perpendicular two-dimensional replica conditions. Additionally, neurites on perpendicular conditions turned to extend along the direction of underlying SC topography. Neurite outgrowth exceeded SC migration in the direction of the underlying anisotropic SC replica after two days in culture. This finding confirms the critical role that SC have in guiding neurite outgrowth and suggests that the mechanism of neurite alignment to SC replicas depends on direct contact with cellular topography. These results suggest that SC topographical replicas may be used to direct and optimize neurite

  2. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    EPA Science Inventory

    There is a need for rapid, efficient and cost effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be...

  3. Cocaine decreases cell survival and inhibits neurite extension of rat locus coeruleus neurons.

    PubMed

    Snow, D M; Smith, J D; Booze, R M; Welch, M A; Mactutus, C F

    2001-01-01

    Cocaine use during pregnancy is affiliated with neurobehavioral abnormalities in offspring that are associated with problems of attention. Given the putative role of the noradrenergic system in attentional processes, impairments in the noradrenergic system may underlie specific attentionally sensitive, neurobehavioral alterations. Recent data using a clinically relevant intravenous (iv) route of administration show that the norepinephrine cell bodies of the locus coeruleus (LC) are a primary target for in utero cocaine exposure. Cell survival and neurite outgrowth of LC neurons were studied using two paradigms: (1) in vitro, using a physiologically relevant concentration of cocaine, and (2) in vivo, using a clinically relevant intravenous rat model. Fetal cocaine exposure significantly decreased neuronal survival (in vitro: P=.0001, n=24; in vivo: P=.0337, n=30), reduced neurite initiation (in vitro: P=.001, n=24; in vivo: P=.0169, n=30), decreased the number of neurites elaborated (in vivo: P=.0031, n=30), and reduced total neurite length (in vivo: P=.0237, n=30). The results of this novel approach toward an understanding of noradrenergic neurons as they respond to cocaine during development suggest that cocaine may affect behavior by negatively regulating neuronal pathfinding and synaptic connectivity. PMID:11418264

  4. Synergic interaction between amyloid precursor protein and neural cell adhesion molecule promotes neurite outgrowth

    PubMed Central

    Chen, Keping; Lu, Huixia; Gao, Tianli; Xue, Xiulei; Wang, Chunling; Miao, Fengqin

    2016-01-01

    Alzheimer's disease (AD) is one of the most common neurodegenerative diseases worldwide. The main features of AD are the pathological changes of density and distribution of intracellular neurofibrillary tangles (NFT) and extracellular amyloid plaques. The processing of amyloid beta precursor protein (APP) to β-amyloid peptide (Aβ) is one of the critical events in the pathogenesis of AD. In this study, we evaluated the role of the interaction of neural cell adhesion molecule (NCAM) and APP in neurite outgrowth using two different experimental systems: PC12E2 cells and hippocampal neurons that were isolated from wild type, APP knock-in and APP knock-out mice. PC12E2 cells or hippocampal neurons were co-cultured with NCAM-negative or NCAM-positive fibroblasts L929 cells. We found that APP promoted neurite outgrowth of PC12E2 cells and hippocampal neurons in either the presence or absence of NCAM. Secreted APP can rescue the neurite outgrowth in hippocampal neurons from APP knock-out mice. The interaction of APP and NCAM had synergic effect in promoting neurite outgrowth in both PC12E2 cells and hippocampal neurons. Our results suggested that the interaction of APP with NCAM played an important role in AD development and therefore could be a potential therapeutic target for AD treatment. PMID:26883101

  5. ACTION OF 50 HZ MAGNETIC FIELDS ON NEURITE OUTGROWTH IN PHEOCHROMOCYTOMA CELLS

    EPA Science Inventory

    This study tests the capacity of 50-Hz magnetic and electric fields to stimulate neurite outgrowth in PC-12D cells, a cell line which originated from a pheochromocytoma in rat adrenal medulla. he cells were plated on collagen-coated plastic petri dishes and exposed to sinusoidal ...

  6. White matter microstructure pathology in classic galactosemia revealed by neurite orientation dispersion and density imaging.

    PubMed

    Timmers, Inge; Zhang, Hui; Bastiani, Matteo; Jansma, Bernadette M; Roebroeck, Alard; Rubio-Gozalbo, M Estela

    2015-03-01

    White matter abnormalities have been observed in patients with classic galactosemia, an inborn error of galactose metabolism. However, magnetic resonance imaging (MRI) data collected in the past were generally qualitative in nature. Our objective was to investigate white matter microstructure pathology and examine correlations with outcome and behaviour in this disease, by using multi-shell diffusion weighted imaging. In addition to standard diffusion tensor imaging (DTI), neurite orientation dispersion and density imaging (NODDI) was used to estimate density and orientation dispersion of neurites in a group of eight patients (aged 16-21 years) and eight healthy controls (aged 15-20 years). Extensive white matter abnormalities were found: neurite density index (NDI) was lower in the patient group in bilateral anterior areas, and orientation dispersion index (ODI) was increased mainly in the left hemisphere. These specific regional profiles are in agreement with the cognitive profile observed in galactosemia, showing higher order cognitive impairments, and language and motor impairments, respectively. Less favourable white matter properties correlated positively with age and age at onset of diet, and negatively with behavioural outcome (e.g. visual working memory). To conclude, this study provides evidence of white matter pathology regarding density and dispersion of neurites in these patients. The results are discussed in light of suggested pathophysiological mechanisms. PMID:25344151

  7. Neurite extension of developing noradrenergic neurons is impaired in genetically epilepsy-prone rats (GEPR-3s): an in vitro study on the locus coeruleus.

    PubMed

    Clough, R W; Peterson, B R; Steenbergen, J L; Jobe, P C; Eells, J B; Browning, R A; Mishra, P K

    1998-01-01

    A primary determinant of seizure susceptibility and severity in genetically epilepsy-prone rats (GEPRs), is a generalized deficiency in the central noradrenergic system of these animals. In particular, this deficiency includes reduced numbers of norepinephrine (NE) synaptic terminals in several brain areas and distinctly fewer NE axons within the auditory tectum. Two strains of GEPRs have been developed: GEPR-3s that have moderately severe clonic seizures and GEPR-9s that have severe tonic seizures culminating in complete hindlimb extension. Seizures in animals of each substrain are preceded by a brief episode of wild running. The developmental profile of NE axonal growth in GEPRs compared to control rats is not known, but may be causally related to NE deficiencies in this seizure model. The present study compared developmental neurite extension of fetal NE neurons in vitro between GEPR-3s and Sprague-Dawley control rats, the strain from which GEPR-3s were originally derived. Neurite arborization of individual NE neurons was assessed by quantitative morphometry following immunocytochemical identification of tyrosine hydroxylase (TH). Preliminary studies using explant and dispersed-cell cultures of control-rat tissues showed that optimal culture parameters to support neuritogenesis of LC neurons included the use of dispersed-cell cultures, Pronectin-F substrate, day-14 gestation donor-tissue, no use of cytosine-arabinofuranoside (ARA-c, a glial mitotic inhibitor) and the presence of co-cultured tectal tissue. Compared to fetal control-rat NE neurons co-cultured with fetal control-rat tectum, NE neurons derived from fetal GEPR-3 LC in co-culture with GEPR-3 tectum exhibited only 30% of the neurite extension of control-rat LC neurons and GEPR-3 LC neurons had a similarly deficient amount of branching. This study suggests, but does not prove, that deficiency in tectal NE in GEPR-3s involves a developmental deficiency in neurite extension from GEPR-3 LC neurons

  8. Electrical excitability of outgrowing neurites of embryonic neurones in cultures of dissociated neural plate of Xenopus laevis.

    PubMed Central

    Willard, A L

    1980-01-01

    1. I have studied the electrical excitability of outgrowing processes of individual neurones in cultures made from dissociated neural plates of embryos of Xenopus laevis prior to the time of neurite outgrowth in vivo. 2. The electrical excitability of neurites was tested by stimulating them extracellularly and recording responses with an intracellular electrode in their cell bodies; neurites were excitable at all times examined. 3. The ionic basis of the excitability of neurites was tested by recording from cells while changing the composition of the salines perfusing the cultures. 4. In cultures less than 10 hr old, all neurites tested made responses which depended on Ca2+. The action potentials of the cell bodies were also Ca2+-dependent at these times. 5. Between 10 and 12 hr in culture, a time at which the cell bodies still made Ca2+-dependent action potentials, neurites acquired the ability to make Na+-dependent responses. At these times, two-thirds of neurites tested retained the ability to produce divalent cation-dependent action potentials when perfused with solutions of isotonic Ba2+. 6. After 12 hr in culture, no neurites were observed to make Ca2+-or Ba2+-dependent responses; only Na+-dependent responses were observed. Cells continued to initiate and elongate new neurites until about 24 hr in culture. Thus neurites sent out at different times in culture differed in their development of excitability. 7. Cell bodies making exclusively Ca2+-dependent action potentials could be found until about 15 hr in culture, after which time a Na+-dependent component appeared. Cell bodies could then be observed to make action potentials which depended on both Ca2+ and Na+ until about 3 days in culture. After 3 days, most cell bodies made predominately Na+-dependent action potentials. Unlike the neurites, cell bodies retained the ability to make action potentials in isotonic Ba2+ for as long as the cultures were maintained (up to 5 days). 8. The possibility that changes

  9. Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis

    EPA Science Inventory

    During development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxicants that interfere with this process may cause in permanent deficits in nervous system function. While many studies have used rodent primary...

  10. Leukemia Inhibitory Factor Induces Neurotransmitter Switching in Transgenic Mice

    NASA Astrophysics Data System (ADS)

    Bamber, Bruce A.; Masters, Brian A.; Hoyle, Gary W.; Brinster, Ralph L.; Palmiter, Richard D.

    1994-08-01

    Leukemia inhibitory factor (LIF) is a cytokine growth factor that induces rat sympathetic neurons to switch their neurotransmitter phenotype from noradrenergic to cholinergic in vitro. To test whether LIF can influence neuronal differentiation in vivo, we generated transgenic mice that expressed LIF in pancreatic islets under the control of the insulin promoter and evaluated the neurotransmitter phenotype of the pancreatic sympathetic innervation. We also used the insulin promoter to coexpress nerve growth factor in the islets, which greatly increased the density of sympathetic innervation and facilitated analysis of the effects of LIF. Our data demonstrate that tyrosine hydroxylase and catecholamines declined and choline acetyltransferase increased in response to LIF. We conclude that LIF can induce neurotransmitter switching of sympathetic neurons in vivo.

  11. Guaifenesin derivatives promote neurite outgrowth and protect diabetic mice from neuropathy.

    PubMed

    Hadimani, Mallinath B; Purohit, Meena K; Vanampally, Chandrashaker; Van der Ploeg, Randy; Arballo, Victor; Morrow, Dwane; Frizzi, Katie E; Calcutt, Nigel A; Fernyhough, Paul; Kotra, Lakshmi P

    2013-06-27

    In diabetic patients, an early index of peripheral neuropathy is the slowing of conduction velocity in large myelinated neurons and a lack of understanding of the basic pathogenic mechanisms hindered therapeutics development. Racemic (R/S)-guaifenesin (1) was identified as a potent enhancer of neurite outgrowth using an in vitro screen. Its R-enantiomer (R)-1 carried the most biological activity, whereas the S-enantiomer (S)-1 was inactive. Focused structural variations to (R/S)-1 was conducted to identify potentially essential groups for the neurite outgrowth activity. In vivo therapeutic studies indicated that both (R/S)-1 and (R)-1 partially prevented motor nerve conduction velocity slowing in a mouse model of type 1 diabetes. In vitro microsomal assays suggested that compounds (R)-1 and (S)-1 are not metabolized rapidly, and PAMPA assay indicated moderate permeability through the membrane. Findings revealed here could lead to the development of novel drugs for diabetic neuropathy. PMID:23758573

  12. Large enhancement in neurite outgrowth on a cell membrane-mimicking conducting polymer.

    PubMed

    Zhu, Bo; Luo, Shyh-Chyang; Zhao, Haichao; Lin, Hsing-An; Sekine, Jun; Nakao, Aiko; Chen, Chi; Yamashita, Yoshiro; Yu, Hsiao-Hua

    2014-01-01

    Although electrically stimulated neurite outgrowth on bioelectronic devices is a promising means of nerve regeneration, immunogenic scar formation can insulate electrodes from targeted cells and tissues, thereby reducing the lifetime of the device. Ideally, an electrode material capable of electrically interfacing with neurons selectively and efficiently would be integrated without being recognized by the immune system and minimize its response. Here we develop a cell membrane-mimicking conducting polymer possessing several attractive features. This polymer displays high resistance towards nonspecific enzyme/cell binding and recognizes targeted cells specifically to allow intimate electrical communication over long periods of time. Its low electrical impedance relays electrical signals efficiently. This material is capable to integrate biochemical and electrical stimulation to promote neural cellular behaviour. Neurite outgrowth is enhanced greatly on this new conducting polymer; in addition, electrically stimulated secretion of proteins from primary Schwann cells can also occur on it. PMID:25060339

  13. Large enhancement in neurite outgrowth on a cell membrane-mimicking conducting polymer

    NASA Astrophysics Data System (ADS)

    Zhu, Bo; Luo, Shyh-Chyang; Zhao, Haichao; Lin, Hsing-An; Sekine, Jun; Nakao, Aiko; Chen, Chi; Yamashita, Yoshiro; Yu, Hsiao-Hua

    2014-07-01

    Although electrically stimulated neurite outgrowth on bioelectronic devices is a promising means of nerve regeneration, immunogenic scar formation can insulate electrodes from targeted cells and tissues, thereby reducing the lifetime of the device. Ideally, an electrode material capable of electrically interfacing with neurons selectively and efficiently would be integrated without being recognized by the immune system and minimize its response. Here we develop a cell membrane-mimicking conducting polymer possessing several attractive features. This polymer displays high resistance towards nonspecific enzyme/cell binding and recognizes targeted cells specifically to allow intimate electrical communication over long periods of time. Its low electrical impedance relays electrical signals efficiently. This material is capable to integrate biochemical and electrical stimulation to promote neural cellular behaviour. Neurite outgrowth is enhanced greatly on this new conducting polymer; in addition, electrically stimulated secretion of proteins from primary Schwann cells can also occur on it.

  14. Anisotropic three-dimensional peptide channels guide neurite outgrowth within a biodegradable hydrogel matrix.

    PubMed

    Musoke-Zawedde, Patricia; Shoichet, Molly S

    2006-09-01

    The objective of this study was to investigate the neurite guidance potential of concentration gradients of glycine-arginine-glycine-aspartic acid-serine (GRGDS) oligopeptides immobilized within three-dimensional patterned cylindrical volumes created in a biodegradable nerve guidance matrix. This was achieved using ultraviolet (UV) laser micropatterning of a hyaluronan (HA) hydrogel matrix modified with S-2-nitrobenzyl cysteine. Upon exposure to focused laser light, the 2-nitrobenzyl group was cleaved, exposing thiol groups which reacted with maleimide-terminated GRGDS exclusively within these laser-defined volumes. We show that the UV laser micropatterning technique can be used to create GRGDS peptide concentration gradients within the oligopeptide channels and that these channels guide neurite outgrowth from primary neural cells. PMID:18458398

  15. The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by ß-amyloid peptide

    PubMed Central

    Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, WE

    2010-01-01

    Background and purpose: β-Amyloid peptide (Aβ) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. Experimental approach: We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Aβ-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Aβ and on neurite outgrowth in PC12 cells were investigated. Key results: Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Aβ1-42. Similar protective effects against Aβ1-42 were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Aβ load was markedly diminished in the brain of those animals after treatment with piracetam. Aβ production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Aβ-induced mitochondrial dysfunction and Aβ-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Conclusion and implications: Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Aβ on brain function. This article is commented on by Moncada, pp. 217–219 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00706.x and to view related papers by Pravdic et al. and Puerta et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00698.x and http://dx.doi.org/10.1111/j

  16. Kalirin is required for BDNF-TrkB stimulated neurite outgrowth and branching.

    PubMed

    Yan, Yan; Eipper, Betty A; Mains, Richard E

    2016-08-01

    Exogenous brain-derived neurotrophic factor (BDNF), acting through TrkB, is known to promote neurite formation and branching. This response to BDNF was eliminated by inhibition of TrkB kinase and by specific inhibition of the GEF1 domain of Kalirin, which activates Rac1. Neurons from Kalrn knockout mice were unable to activate Rac1 in response to BDNF. BDNF-triggered neurite outgrowth was abolished when Kalrn expression was reduced using shRNA that targets all of the major Kalrn isoforms, and reduced in neurons from Kalrn knockout mice. The Kalrn isoforms expressed early in development also include a GEF2 domain that activates RhoA. However, BDNF-stimulated neurite outgrowth in Kalrn knockout neurons was rescued by expression of Kalirin-7, which includes only the GEF1 domain but lacks the GEF2 domain. Dendritic morphogenesis, which requires spatially restricted, coordinated changes in the actin cytoskeleton and in the organization of microtubules, involves essential contributions from multiple Rho GEFs. Since Tiam1, another Rho GEF, is also required for BDNF-stimulated neurite outgrowth, an inhibitory fragment of Tiam1 (PHn-CC-EX) was tested and found to interfere with both Kalirin and Tiam1 GEF activity. The prolonged TrkB activation observed in response to BDNF in Kalrn knockout neurons and the altered time course and extent of ERK, CREB and Akt activation observed in the absence of Kalrn would be expected to alter the response of these neurons to other regulatory factors. PMID:27036892

  17. The role of cell adhesion molecules in visual circuit formation: from neurite outgrowth to maps and synaptic specificity.

    PubMed

    Missaire, Mégane; Hindges, Robert

    2015-06-01

    The formation of visual circuitry is a multistep process that involves cell-cell interactions based on a range of molecular mechanisms. The correct implementation of individual events, including axon outgrowth and guidance, the formation of the topographic map, or the synaptic targeting of specific cellular subtypes, are prerequisites for a fully functional visual system that is able to appropriately process the information captured by the eyes. Cell adhesion molecules (CAMs) with their adhesive properties and their high functional diversity have been identified as key actors in several of these fundamental processes. Because of their growth-promoting properties, CAMs play an important role in neuritogenesis. Furthermore, they are necessary to control additional neurite development, regulating dendritic spacing and axon pathfinding. Finally, trans-synaptic interactions of CAMs ensure cell type-specific connectivity as a basis for the establishment of circuits processing distinct visual features. Recent discoveries implicating CAMs in novel mechanisms have led to a better general understanding of neural circuit formation, but also revealed an increasing complexity of their function. This review aims at describing the different levels of action for CAMs to shape neural connectivity, with a special focus on the visual system. PMID:25649254

  18. Human Schwann Cells Seeded on a Novel Collagen-Based Microstructured Nerve Guide Survive, Proliferate, and Modify Neurite Outgrowth

    PubMed Central

    van Neerven, Sabien G. A.; Haastert-Talini, Kirsten; Tolba, René H.; Pallua, Norbert; Bozkurt, Ahmet

    2014-01-01

    A variety of new bioartificial nerve guides have been tested preclinically for their safety and nerve regeneration supporting properties. So far, only a limited number of biomaterials have been tested in humans since the step from preclinical work to a clinical application is challenging. We here present an in vitro model with human Schwann cells (hSCs) as an intermediate step towards clinical application of the nerve guide Perimaix, a collagen-based microstructured 3D scaffold containing numerous longitudinal guidance channels for directed axonal growth. hSCs were seeded onto different prototypes of Perimaix and cultivated for 14 days. hSC adhered to the scaffold, proliferated, and demonstrated healthy Schwann cell morphology (spindle shaped cell bodies, bipolar oriented processes) not only at the surface of the material, but also in the deeper layers of the scaffold. The general well-being of the cells was quantitatively confirmed by low levels of lactate dehydrogenase release into the culture medium. Moreover, conditioned medium of hSCs that were cultivated on Perimaix was able to modify neurite outgrowth from sensory dorsal root ganglion neurons. Overall these data indicate that Perimaix is able to provide a matrix that can promote the attachment and supports process extension, migration, and proliferation of hSC. PMID:24895582

  19. Age-dependent differences in brain tissue microstructure assessed with neurite orientation dispersion and density imaging.

    PubMed

    Merluzzi, Andrew P; Dean, Douglas C; Adluru, Nagesh; Suryawanshi, Gaurav S; Okonkwo, Ozioma C; Oh, Jennifer M; Hermann, Bruce P; Sager, Mark A; Asthana, Sanjay; Zhang, Hui; Johnson, Sterling C; Alexander, Andrew L; Bendlin, Barbara B

    2016-07-01

    Human aging is accompanied by progressive changes in executive function and memory, but the biological mechanisms underlying these phenomena are not fully understood. Using neurite orientation dispersion and density imaging, we sought to examine the relationship between age, cellular microstructure, and neuropsychological scores in 116 late middle-aged, cognitively asymptomatic participants. Results revealed widespread increases in the volume fraction of isotropic diffusion and localized decreases in neurite density in frontal white matter regions with increasing age. In addition, several of these microstructural alterations were associated with poorer performance on tests of memory and executive function. These results suggest that neurite orientation dispersion and density imaging is capable of measuring age-related brain changes and the neural correlates of poorer performance on tests of cognitive functioning, largely in accordance with published histological findings and brain-imaging studies of people of this age range. Ultimately, this study sheds light on the processes underlying normal brain development in adulthood, knowledge that is critical for differentiating healthy aging from changes associated with dementia. PMID:27255817

  20. Automatic Robust Neurite Detection and Morphological Analysis of Neuronal Cell Cultures in High-content Screening

    PubMed Central

    Wu, Chaohong; Schulte, Joost; Sepp, Katharine J.; Littleton, J. Troy

    2011-01-01

    Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutamine-mediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington’s Disease (HD) model. PMID:20405243

  1. Neuroprotective effects of ginsenoside Rb1 on hippocampal neuronal injury and neurite outgrowth

    PubMed Central

    Liu, Juan; He, Jing; Huang, Liang; Dou, Ling; Wu, Shuang; Yuan, Qionglan

    2014-01-01

    Ginsenoside Rb1 has been reported to exert anti-aging and anti-neurodegenerative effects. In the present study, we investigate whether ginsenoside Rb1 is involved in neurite outgrowth and neuroprotection against damage induced by amyloid beta (25–35) in cultured hippocampal neurons, and explore the underlying mechanisms. Ginsenoside Rb1 significantly increased neurite outgrowth in hippocampal neurons, and increased the expression of phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2. These effects were abrogated by API-2 and PD98059, inhibitors of the signaling proteins Akt and MEK. Additionally, cultured hippocampal neurons were exposed to amyloid beta (25–35) for 30 minutes; ginsenoside Rb1 prevented apoptosis induced by amyloid beta (25–35), and this effect was blocked by API-2 and PD98059. Furthermore, ginsenoside Rb1 significantly reversed the reduction in phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2 levels induced by amyloid beta (25–35), and API-2 neutralized the effect of ginsenoside Rb1. The present results indicate that ginsenoside Rb1 enhances neurite outgrowth and protects against neurotoxicity induced by amyloid beta (25–35) via a mechanism involving Akt and extracellular signal-regulated kinase 1/2 signaling. PMID:25206916

  2. Quantifying Spiral Ganglion Neurite and Schwann Behavior on Micropatterned Polymer Substrates.

    PubMed

    Cheng, Elise L; Leigh, Braden; Guymon, C Allan; Hansen, Marlan R

    2016-01-01

    The first successful in vitro experiments on the cochlea were conducted in 1928 by Honor Fell (Fell, Arch Exp Zellforsch 7(1):69-81, 1928). Since then, techniques for culture of this tissue have been refined, and dissociated primary culture of the spiral ganglion has become a widely accepted in vitro model for studying nerve damage and regeneration in the cochlea. Additionally, patterned substrates have been developed that facilitate and direct neural outgrowth. A number of automated and semi-automated methods for quantifying this neurite outgrowth have been utilized in recent years (Zhang et al., J Neurosci Methods 160(1):149-162, 2007; Tapias et al., Neurobiol Dis 54:158-168, 2013). Here, we describe a method to study the effect of topographical cues on spiral ganglion neurite and Schwann cell alignment. We discuss our microfabrication process, characterization of pattern features, cell culture techniques for both spiral ganglion neurons and spiral ganglion Schwann cells. In addition, we describe protocols for reducing fibroblast count, immunocytochemistry, and methods for quantifying neurite and Schwann cell alignment. PMID:27259935

  3. MicroRNA-320 Induces Neurite Outgrowth by Targeting ARPP-1

    PubMed Central

    White, Robin E.; Giffard, Rona G.

    2012-01-01

    MicroRNAs are important in central nervous system development, functioning, and pathophysiology. Here we demonstrate that increasing levels of microRNA 320 (miR-320) for 3 days markedly increases neurite length and at 4 days reduces total cell number in N2A cells. In silico analysis of possible miR-320 targets identified cAMP-regulated phosphoprotein-19 kDa (ARPP-19) and semaphorin 3a (Sema3a) as potential targets that could be involved. ARPP-19 was validated by demonstrating reduced mRNA and protein levels when miR-320 was overexpressed, while miR-320 had no effect on Sema3a expression. ARPP-19 is known to inhibit protein phosphatase-2A (PP2A) activity, which inhibits mitosis and induces neurite outgrowth, making this the likely mechanism. Thus increased levels of miR-320 leads to decreased levels of ARPP-19, increased neurite length, and fewer total cells. These data suggest that miR-320 could play a role in neuronal development and might be a target to enhance neuronal regeneration following injury. PMID:22617447

  4. Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

    PubMed Central

    Koch, J C; Bitow, F; Haack, J; d'Hedouville, Z; Zhang, J-N; Tönges, L; Michel, U; Oliveira, L M A; Jovin, T M; Liman, J; Tatenhorst, L; Bähr, M; Lingor, P

    2015-01-01

    Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered. PMID:26158517

  5. Mycolactone-mediated neurite degeneration and functional effects in cultured human and rat DRG neurons

    PubMed Central

    Sinisi, M; Fox, M; MacQuillan, A; Quick, T; Korchev, Y; Bountra, C; McCarthy, T; Anand, P

    2016-01-01

    Background Mycolactone is a polyketide toxin secreted by the mycobacterium Mycobacterium ulcerans, responsible for the extensive hypoalgesic skin lesions characteristic of patients with Buruli ulcer. A recent pre-clinical study proposed that mycolactone may produce analgesia via activation of the angiotensin II type 2 receptor (AT2R). In contrast, AT2R antagonist EMA401 has shown analgesic efficacy in animal models and clinical trials for neuropathic pain. We therefore investigated the morphological and functional effects of mycolactone in cultured human and rat dorsal root ganglia (DRG) neurons and the role of AT2R using EMA401. Primary sensory neurons were prepared from avulsed cervical human DRG and rat DRG; 24 h after plating, neurons were incubated for 24 to 96 h with synthetic mycolactone A/B, followed by immunostaining with antibodies to PGP9.5, Gap43, β tubulin, or Mitotracker dye staining. Acute functional effects were examined by measuring capsaicin responses with calcium imaging in DRG neuronal cultures treated with mycolactone. Results Morphological effects: Mycolactone-treated cultures showed dramatically reduced numbers of surviving neurons and non-neuronal cells, reduced Gap43 and β tubulin expression, degenerating neurites and reduced cell body diameter, compared with controls. Dose-related reduction of neurite length was observed in mycolactone-treated cultures. Mitochondria were distributed throughout the length of neurites and soma of control neurons, but clustered in the neurites and soma of mycolactone-treated neurons. Functional effects: Mycolactone-treated human and rat DRG neurons showed dose-related inhibition of capsaicin responses, which were reversed by calcineurin inhibitor cyclosporine and phosphodiesterase inhibitor 3-isobutyl-1-Methylxanthine, indicating involvement of cAMP/ATP reduction. The morphological and functional effects of mycolactone were not altered by Angiotensin II or AT2R antagonist EMA401. Conclusion Mycolactone

  6. Tumor necrosis factor induced stimulation of granulopoiesis and radioprotection.

    PubMed

    Urbaschek, R; Männel, D N; Urbaschek, B

    1987-01-01

    Human recombinant tumor necrosis factor, TNF, was used to assess its ability to stimulate granulopoiesis and to protect mice against lethal irradiation, effects known to be inducable with TNF-rich postendotoxin serum from BCG infected mice (BCG/ET serum). Although the endotoxin contamination of this TNF preparation is extremely low its effects were compared in endotoxin low responder C3H/HeJ mice and susceptible NMRI mice. TNF is a potent inducer of serum colony stimulating activity, CSA, in both mouse strains. In peripheral blood a marked granulocytosis with a concomitant decrease in lymphocytes and monocytopenia occurs at 2 hours after injection of TNF. Moreover, TNF induces an increase in the number of splenic myelopoietic committed stem cells (GM-CFC, granulocyte-macrophage colony forming cells) determined five days after injection. The lethality rate, registered over 30 days after exposure to 660 cGy whole body X-irradiation is reduced to 40% in C3H/HeJ mice as compared to 75% in control animals. The reduction in lethality is observed both, when TNF was injected 24 hours before or after irradiation. In vitro, TNF significantly increases the number of colonies in the presence of CSA in bone marrow cultures. TNF per se does not effect colony growth. The studies reported here demonstrate that TNF is a myelopoiesis stimulating factor in mice which may be related to the reduction in lethality following whole body irradiation. PMID:3306175

  7. Synthesis and neurite growth evaluation of new analogues of honokiol, a neolignan with potent neurotrophic activity.

    PubMed

    Praveen Kumar, Vemula; Gajendra Reddy, R; Vo, Duc Duy; Chakravarty, Sumana; Chandrasekhar, Srivari; Grée, René

    2012-02-01

    A versatile synthetic route is reported towards the preparation of new analogues for potent neurotrophic agent biaryl-type lignan honokiol. A focused 24-membered library of derivatives containing five different groups at 5'-position of honokiol has been prepared in fair to good overall yields. Compared to the natural product, or to analogues with a short alkyl chain in this position, these new derivatives have lost most of the neurotrophic activity. PMID:22209461

  8. The Susd2 protein regulates neurite growth and excitatory synaptic density in hippocampal cultures.

    PubMed

    Nadjar, Yann; Triller, Antoine; Bessereau, Jean-Louis; Dumoulin, Andrea

    2015-03-01

    Complement control protein (CCP) domains have adhesion properties and are commonly found in proteins that control the complement immune system. However, an increasing number of proteins containing CCP domains have been reported to display neuronal functions. Susd2 is a transmembrane protein containing one CCP domain. It was previously identified as a tumor-reversing protein, but has no characterized function in the CNS. The present study investigates the expression and function of Susd2 in the rat hippocampus. Characterization of Susd2 during development showed a peak in mRNA expression two weeks after birth. In hippocampal neuronal cultures, the same expression profile was observed at 15days in vitro for both mRNA and protein, a time consistent with synaptogenesis in our model. At the subcellular level, Susd2 was located on the soma, axons and dendrites, and appeared to associate preferentially with excitatory synapses. Inhibition of Susd2 by shRNAs led to decreased numbers of excitatory synaptic profiles, exclusively. Also, morphological parameters were studied on young (5DIV) developing neurons. After Susd2 inhibition, an increase in dendritic tree length but a decrease in axon elongation were observed, suggesting changes in adhesion properties. Our results demonstrate a dual role for Susd2 at different developmental stages, and raise the question whether Susd2 and other CCP-containing proteins expressed in the CNS could be function-related. PMID:25724483

  9. Fabrication of Aligned Conducting PPy-PLLA Fiber Films and Their Electrically Controlled Guidance and Orientation for Neurites.

    PubMed

    Zou, Yuanwen; Qin, Jiabang; Huang, Zhongbing; Yin, Guangfu; Pu, Ximing; He, Da

    2016-05-25

    Electrically conductive biomaterial scaffolds have great potential in neural tissue regeneration. In this work, an aligned conductive fibrous scaffold was prepared by electrospinning PLLA on rotating collector and chemical oxidation polymerization of pyrrole (PPy) codoped with poly(glutamic acid)/dodecyl benzenesulfonic acid sodium. The characterization results of composition, structure and mechanics of fiber films show that the existence of weak polar van der Waals' force between PPy coating and PLLA fibers. The resistivity of aligned rough PPy-PLLA fiber film (about 800 nm of fiber diameter) at the perpendicular and parallel directions is 0.971 and 0.874 Ω m, respectively. Aligned rough PPy-PLLA fiber film could guide the extension of 68% PC12 neurites along the direction of fiber axis. Under electrostimulation (ES) of 100, 200, and 400 mV/cm, median neurite lengths of differentiated PC12 on aligned fiber-films are 128, 149, and 141 μm, respectively. Furthermore, under ES of 100, 200, and 400 mV/cm, the alignment rate of neurite along the electropotential direction (angle between neurite and electropotential direction ≤10°) on random fibers film are 17, 23, and 28%, respectively, and the alignment rate of neurites along the fiber axis (angle between neurite and fiber axis ≤10°) on aligned fibers film reach to 76, 83, and 79%, respectively, indicating that the combination of ES and rough conducting aligned structure could adjust the alignment of cellular neurites along the direction of the fiber axis or electropotential. PMID:27172537

  10. Downregulation of the Ras–Mitogen-Activated Protein Kinase Pathway by the EphB2 Receptor Tyrosine Kinase Is Required for Ephrin-Induced Neurite Retraction

    PubMed Central

    Elowe, Sabine; Holland, Sacha J.; Kulkarni, Sarang; Pawson, Tony

    2001-01-01

    Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones. PMID:11585923

  11. Sialylation of neurites inhibits complement-mediated macrophage removal in a human macrophage-neuron Co-Culture System.

    PubMed

    Linnartz-Gerlach, Bettina; Schuy, Christine; Shahraz, Anahita; Tenner, Andrea J; Neumann, Harald

    2016-01-01

    The complement system has been implicated in the removal of dysfunctional synapses and neurites during development and in disease processes in the mouse, but it is unclear how far the mouse data can be transferred to humans. Here, we co-cultured macrophages derived from human THP1 monocytes and neurons derived from human induced pluripotent stem cells, to study the role of the complement system in a human model. Components of the complement system were expressed by the human macrophages and human neuronal culture, while receptors of the complement cascade were expressed by human macrophages as shown via gene transcript analysis and flow cytometry. We mimicked pathological conditions leading to an altered glycocalyx by treatment of human neurons with sialidases. Desialylated human neurites were opsonized by the complement component C1q. Furthermore, human neurites with an intact sialic acid cap remained untouched, while desialylated human neurites were removed and ingested by human macrophages. While blockage of the complement receptor 1 (CD35) had no effect, blockage of CD11b as part of the complement receptor 3 (CR3) reversed the effect on macrophage phagocytosis of desialylated human neurites. Data demonstrate that in the human system sialylation of the neuronal glycocalyx serves as an inhibitory flag for complement binding and CR3-mediated phagocytosis by macrophages. PMID:26257016

  12. Suppression of Radixin and Moesin Alters Growth Cone Morphology, Motility, and Process Formation In Primary Cultured Neurons

    PubMed Central

    Paglini, Gabriela; Kunda, Patricia; Quiroga, Santiago; Kosik, Kenneth; Cáceres, Alfredo

    1998-01-01

    In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin–radixin or ezrin–moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin–moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8–10 times slower than the one of growth cones from control neurons. Radixin–moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phenomenon that appears to be directly linked with the alterations in growth cone structure and motility. Taken collectively, our data suggest that by regulating key aspects of growth cone development and maintenance, radixin and moesin modulate neurite formation and the development of neuronal polarity. PMID:9786954

  13. Organic Photovoltaics and Bioelectrodes Providing Electrical Stimulation for PC12 Cell Differentiation and Neurite Outgrowth.

    PubMed

    Hsiao, Yu-Sheng; Liao, Yan-Hao; Chen, Huan-Lin; Chen, Peilin; Chen, Fang-Chung

    2016-04-13

    Current bioelectronic medicines for neurological therapies generally involve treatment with a bioelectronic system comprising a power supply unit and a bioelectrode device. Further integration of wireless and self-powered units is of practical importance for implantable bioelectronics. In this study, we developed biocompatible organic photovoltaics (OPVs) for serving as wireless electrical power supply units that can be operated under illumination with near-infrared (NIR) light, and organic bioelectronic interface (OBEI) electrode devices as neural stimulation electrodes. The OPV/OBEI integrated system is capable to provide electrical stimulation (ES) as a means of enhancing neuron-like PC12 cell differentiation and neurite outgrowth. For the OPV design, we prepared devices incorporating two photoactive material systems--β-carotene/N,N'-dioctyl-3,4,9,10-perylenedicarboximide (β-carotene/PTCDI-C8) and poly(3-hexylthiophene)/phenyl-C61-butyric acid methyl ester (P3HT/PCBM)--that exhibited open circuit voltages of 0.11 and 0.49 V, respectively, under NIR light LED (NLED) illumination. Then, we connected OBEI devices with different electrode gaps, incorporating biocompatible poly(hydroxymethylated-3,4-ethylenedioxythiophene), to OPVs to precisely tailor the direct current electric field conditions during the culturing of PC12 cells. This NIR light-driven OPV/OBEI system could be engineered to provide tunable control over the electric field (from 220 to 980 mV mm(-1)) to promote 64% enhancement in the neurite length, direct the neurite orientation on chips, or both. The OPV/OBEI integrated systems under NIR illumination appear to function as effective power delivery platforms that should meet the requirements for wirelessly offering medical ES to a portion of the nervous system; they might also be a key technology for the development of next-generation implantable bioelectronics. PMID:26999636

  14. A subset of chicken statoacoustic ganglion neurites are repelled by Slit1 and Slit2

    PubMed Central

    Battisti, Andrea C.; Fantetti, Kristen N.; Moyers, Bryan A.; Fekete, Donna M.

    2014-01-01

    Mechanosensory hair cells in the chicken inner ear are innervated by bipolar afferent neurons of the statoacoustic ganglion (SAG). During development, individual SAG neurons project their peripheral process to only one of eight distinct sensory organs. These neuronal subtypes may respond differently to guidance cues as they explore the periphery in search of their target. Previous gene expression data suggested that Slit repellants might channel SAG neurites into the sensory primordia, based on the presence of robo transcripts in the neurons and the confinement of slit transcripts to the flanks of the prosensory domains. This led to the prediction that excess Slit proteins would impede the outgrowth of SAG neurites. As predicted, axonal projections to the primordium of the anterior crista were reduced 2-3 days after electroporation of either slit1 or slit2 expression plasmids into the anterior pole of the otocyst on embryonic day 3 (E3). The posterior crista afferents, which normally grow through and adjacent to slit expression domains as they are navigating towards the posterior pole of the otocyst, did not show Slit responsiveness when similarly challenged by ectopic delivery of slit to their targets. The sensitivity to ectopic Slits shown by the anterior crista afferents was more the exception than the rule: responsiveness to Slits was not observed when the entire E4 SAG was challenged with Slits for 40 hours in vitro. The corona of neurites emanating from SAG explants was unaffected by the presence of purified human Slit1 and Slit2 in the culture medium. Reduced axon outgrowth from E8 olfactory bulbs cultured under similar conditions for 24 hours confirmed bioactivity of purified human Slits on chicken neurons. In summary, differential sensitivity to Slit repellents may influence the directional outgrowth of otic axons toward either the anterior or posterior otocyst. PMID:24456709

  15. Functional Consequences of Neurite Orientation Dispersion and Density in Humans across the Adult Lifespan

    PubMed Central

    Nazeri, Arash; Chakravarty, M. Mallar; Rotenberg, David J.; Rajji, Tarek K.; Rathi, Yogesh; Michailovich, Oleg V.

    2015-01-01

    As humans age, a characteristic pattern of widespread neocortical dendritic disruption coupled with compensatory effects in hippocampus and other subcortical structures is shown in postmortem investigations. It is now possible to address age-related effects on gray matter (GM) neuritic organization and density in humans using multishell diffusion-weighted MRI and the neurite-orientation dispersion and density imaging (NODDI) model. In 45 healthy individuals across the adult lifespan (21–84 years), we used a multishell diffusion imaging and the NODDI model to assess the intraneurite volume fraction and neurite orientation-dispersion index (ODI) in GM tissues. We also determined the functional correlates of variations in GM microstructure by obtaining resting-state fMRI and behavioral data. We found a significant age-related deficit in neocortical ODI (most prominently in frontoparietal regions), whereas increased ODI was observed in hippocampus and cerebellum with advancing age. Neocortical ODI outperformed cortical thickness and white matter fractional anisotropy for the prediction of chronological age in the same individuals. Higher GM ODI sampled from resting-state networks with known age-related susceptibility (default mode and visual association networks) was associated with increased functional connectivity of these networks, whereas the task-positive networks tended to show no association or even decreased connectivity. Frontal pole ODI mediated the negative relationship of age with executive function, whereas hippocampal ODI mediated the positive relationship of age with executive function. Our in vivo findings align very closely with the postmortem data and provide evidence for vulnerability and compensatory neural mechanisms of aging in GM microstructure that have functional and cognitive impact in vivo. PMID:25632148

  16. β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells

    PubMed Central

    Girón, María D.; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M.

    2015-01-01

    β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth. PMID:26267903

  17. Neurite Fasciculation Mediated by Complexes of Axonin-1 and Ng Cell Adhesion Molecule

    PubMed Central

    Kunz, Stefan; Spirig, Marianne; Ginsburg, Claudia; Buchstaller, Andrea; Berger, Philipp; Lanz, Rainer; Rader, Christoph; Vogt, Lorenz; Kunz, Beat; Sonderegger, Peter

    1998-01-01

    Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1–NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM–NgCAM interaction could be established simultaneously with the axonin-1–NgCAM interaction. In contrast, the axonin-1–NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons. PMID:9852159

  18. Chroman-like cyclic prenylflavonoids promote neuronal differentiation and neurite outgrowth and are neuroprotective.

    PubMed

    Oberbauer, Eleni; Urmann, Corinna; Steffenhagen, Carolin; Bieler, Lara; Brunner, Doris; Furtner, Tanja; Humpel, Christian; Bäumer, Bastian; Bandtlow, Christine; Couillard-Despres, Sebastien; Rivera, Francisco J; Riepl, Herbert; Aigner, Ludwig

    2013-11-01

    Flavonoids target a variety of pathophysiological mechanisms and are therefore increasingly considered as compounds encompassed with therapeutic potentials in diseases such as cancer, diabetes, arteriosclerosis, and neurodegenerative diseases and mood disorders. Hops (Humulus lupulus L.) is rich in flavonoids such as the flavanone 8-prenylnaringenin, which is the most potent phytoestrogen identified so far, and the prenylchalcone xanthohumol, which has potent tumor-preventive, anti-inflammatory and antiviral activities. In the present study, we questioned whether hops-derived prenylflavonoids and synthetic derivatives thereof act on neuronal precursor cells and neuronal cell lines to induce neuronal differentiation, neurite outgrowth and neuroprotection. Therefore, mouse embryonic forebrain-derived neural precursors and Neuro2a neuroblastoma-derived cells were stimulated with the prenylflavonoids of interest, and their potential to activate the promoter of the neuronal fate-specific doublecortin gene and to stimulate neuronal differentiation and neurite outgrowth was analyzed. In this screening, we identified highly "neuroactive" compounds, which we termed "enhancement of neuronal differentiation factors" (ENDFs). The most potent molecule, ENDF1, was demonstrated to promote neuronal differentiation of neural stem cells and neurite outgrowth of cultured dorsal root ganglion neurons and protected neuronal PC12 cells from cobalt chloride-induced as well as cholinergic neurons of the nucleus basalis of Meynert from deafferentation-induced cell death. The results indicate that hops-derived prenylflavonoids such as ENDFs might be powerful molecules to promote neurogenesis, neuroregeneration and neuroprotection in cases of chronic neurodegenerative diseases, acute brain and spinal cord lesion and age-associated cognitive impairments. PMID:24070601

  19. Two new dendrocandins with neurite outgrowth-promoting activity from Dendrobium officinale.

    PubMed

    Yang, Liu; Liu, Shou-Jin; Luo, Huai-Rong; Cui, Juan; Zhou, Jun; Wang, Xuan-Jun; Sheng, Jun; Hu, Jiang-Miao

    2015-01-01

    Two new bibenzyl derivatives, dendrocandin T (1) and dendrocandin U (2), together with eight known bibenzyls, were isolated from the stems of Dendrobium officinale. Those compounds were sent for the first time for central nervous system-related bioassay and the results indicated that compounds 3, 4, and 5 have a certain degree of neurite outgrowth-promoting activity, and compounds 1, 2, 6, and 7 also have weak activity. The results indicated that D. officinale used as health food and traditional Chinese medicine "Tiepi Shihu" has a health function of neurotrophic effects. PMID:25289696

  20. Angiotensin II AT2 receptors regulate NGF-mediated neurite outgrowth via the NO-cGMP pathway.

    PubMed

    Hashikawa-Hobara, Narumi; Hashikawa, Naoya

    2016-09-16

    We investigated whether Angiotensin II type 2 (AT2) receptor activation was involved in NGF-induced nerve regeneration. NGF-mediated neurite outgrowth in cultured dorsal root ganglia (DRG) cells was significantly inhibited by AT2 receptor antagonist (PD123,319) treatment. AT2 receptor knockdown also inhibited NGF-mediated neurite outgrowth. To determine the mechanisms, we analyzed the NO-cGMP pathway. The cGMP analog increased NGF-mediated nerve elongation, which inhibited by PD123,319. Furthermore, soluble guanylate cyclase expression was significantly less in NGF and PD123,319 treatment DRG than in NGF treatment alone. These results suggest that NGF-mediated neurite outgrowth is suppressed by AT2 receptor signaling via the NO-cGMP-PKG pathway. PMID:27524238

  1. Slit1 promotes regenerative neurite outgrowth of adult dorsal root ganglion neurons in vitro via binding to the Robo receptor.

    PubMed

    Zhang, Hai Ying; Zheng, Lin Feng; Yi, Xi Nan; Chen, Zhi Bin; He, Zhong Ping; Zhao, Dan; Zhang, Xian Fang; Ma, Zhi Jian

    2010-07-01

    Secreted Slit proteins have previously been shown to signal through Roundabout (Robo) receptors to negatively regulate axon guidance and cell migration. During vertebrate development, Slit proteins have also been shown to stimulate branching and elongation of sensory axons and cortical dendrites. In this study, Slit1/Robo2 mRNA and protein expressions were detected in adult rat dorsal root ganglion (DRG) and in cultured DRG neurons. Treatment of both models with recombinant, soluble Slit1 protein was found to promote neurite outgrowth and elongation. In contrast, treatment with a recombinant human Robo2/Fc chimera inhibited neurite outgrowth and elongation. When adult DRG and cultured DRG neurons were pretreated with soluble recombinant human Robo2/Fc chimera, neurite outgrowth and elongation was not induced. These findings indicate that Slit1/Robo2 signaling may have a role in regulating peripheral nerve regeneration. PMID:20172023

  2. Embryonic substantia nigra grafts in the mesencephalon send neurites to the host striatum in non-human primate after overexpression of GDNF

    PubMed Central

    Redmond, D.E.; Elsworth, J.D.; Roth, R.H.; Leranth, C.; Collier, T.J.; Blanchard, B.; Bjugstad, K.B.; Samulski, R.J.; Aebischer, P.; Sladek, J.R.

    2010-01-01

    In spite of partial success in treating Parkinson's disease using ectopically placed grafts of dopamine-producing cells, restoration of the original neuroanatomical circuits, if possible, might work better. Previous evidence of normal anatomic projections from ventral mesencephalic (VM) grafts placed in the substantia nigra (SN) has been limited to neonatal rodents and double grafting or bridging procedures. This study attempted to determine whether injection of a potent growth promoting factor, glial cell line-derived neurotrophic factor (GDNF), into the target regions or placement of fetal striatal co-grafts in the nigrostriatal pathway might elicit neuritic outgrowth to the caudate nucleus. Four adult St. Kitts green monkeys received embryonic VM grafts into the rostral mesencephalon near the host substantia nigra, and injections of AAV2/GDNF or EIAV/GDNF into the caudate. Three adult monkeys were co-grafted with fetal VM tissue near the substantia nigra and fetal striatal grafts (STR) 2.5 mm rostral in the nigrostriatal pathway. Before sacrifice, the striatal target regions were injected with the retrograde tracer fluorogold (FG). FG label was found in tyrosine hydroxylase-labeled neurons in VM grafts in the SN of only those monkeys that received AAV2/GDNF vector injections into the ipsilateral striatum. All monkeys showed FG labeling in the host substantia nigra when FG labeling was injected on the same side. These data show that grafted dopaminergic neurons can extend neurites to a distant target releasing an elevated concentration of GDNF, and suggest that grafted neurons can be placed into appropriate loci for potential tract reconstruction. PMID:19399891

  3. The Effect of Surface Modification of Aligned Poly-L-Lactic Acid Electrospun Fibers on Fiber Degradation and Neurite Extension

    PubMed Central

    Schaub, Nicholas J.; Le Beux, Clémentine; Miao, Jianjun; Linhardt, Robert J.; Alauzun, Johan G.; Laurencin, Danielle; Gilbert, Ryan J.

    2015-01-01

    The surface of aligned, electrospun poly-L-lactic acid (PLLA) fibers was chemically modified to determine if surface chemistry and hydrophilicity could improve neurite extension from chick dorsal root ganglia. Specifically, diethylenetriamine (DTA, for amine functionalization), 2-(2-aminoethoxy)ethanol (AEO, for alcohol functionalization), or GRGDS (cell adhesion peptide) were covalently attached to the surface of electrospun fibers. Water contact angle measurements revealed that surface modification of electrospun fibers significantly improved fiber hydrophilicity compared to unmodified fibers (p < 0.05). Scanning electron microscopy (SEM) of fibers revealed that surface modification changed fiber topography modestly, with DTA modified fibers displaying the roughest surface structure. Degradation of chemically modified fibers revealed no change in fiber diameter in any group over a period of seven days. Unexpectedly, neurites from chick DRG were longest on fibers without surface modification (1651 ± 488 μm) and fibers containing GRGDS (1560 ± 107 μm). Fibers modified with oxygen plasma (1240 ± 143 μm) or DTA (1118 ± 82 μm) produced shorter neurites than the GRGDS or unmodified fibers, but were not statistically shorter than unmodified and GRGDS modified fibers. Fibers modified with AEO (844 ± 151 μm) were significantly shorter than unmodified and GRGDS modified fibers (p<0.05). Based on these results, we conclude that fiber hydrophilic enhancement alone on electrospun PLLA fibers does not enhance neurite outgrowth. Further work must be conducted to better understand why neurite extension was not improved on more hydrophilic fibers, but the results presented here do not recommend hydrophilic surface modification for the purpose of improving neurite extension unless a bioactive ligand is used. PMID:26340351

  4. Effect of Cell Adhesion Molecules on the Neurite Outgrowth of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons.

    PubMed

    Peng, Su-Ping; Schachner, Melitta; Boddeke, Erik; Copray, Sjef

    2016-04-01

    Intrastriatal transplantation of dopaminergic neurons has been shown to be a potentially very effective therapeutic approach for the treatment of Parkinson's disease (PD). With the detection of induced pluripotent stem cells (iPSCs), an unlimited source of autologous dopaminergic (DA) neurons became available. Although the iPSC-derived dopaminergic neurons exhibited most of the fundamental dopaminergic characteristics, detailed analysis and comparison with primary DA neurons have shown some aberrations in the expression of genes involved in neuronal development and neurite outgrowth. The limited outgrowth of the iPSC-derived DA neurons may hamper their potential application in cell transplantation therapy for PD. In the present study, we examined whether the forced expression of L1 cell adhesion molecule (L1CAM) and polysialylated neuronal cell adhesion molecule (PSA-NCAM), via gene transduction, can promote the neurite formation and outgrowth of iPSC-derived DA neurons. In cultures on astrocyte layers, both adhesion factors significantly increased neurite formation of the adhesion factor overexpressing iPSC-derived DA neurons in comparison to control iPSC-derived DA neurons. The same tendency was observed when the DA neurons were plated on postnatal organotypic striatal slices; however, this effect did not reach statistical significance. Next, we examined the neurite outgrowth of the L1CAM- or PSA-NCAM-overexpressing iPSC-derived DA neurons after implantation in the striatum of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats, the animal model for PD. Like the outgrowth on the organotypic striatal slices, no significant L1CAM- and PSA-NCAM-enforced neurite outgrowth of the implanted DA neurons was observed. Apparently, induced expression of L1CAM or PSA-NCAM in the iPSC-derived DA neurons cannot completely restore the neurite outgrowth potential that was reduced in these DA neurons as a consequence of epigenetic aberrations resulting from the i

  5. Expression of a Soluble Isoform of Cell Adhesion Molecule 1 in the Brain and Its Involvement in Directional Neurite Outgrowth

    PubMed Central

    Hagiyama, Man; Ichiyanagi, Naoki; Kimura, Keiko B.; Murakami, Yoshinori; Ito, Akihiko

    2009-01-01

    Cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, is expressed on superior cervical ganglion neurites and mediates cell–cell adhesion by trans-homophilic binding. In addition to the membrane-bound form, we have previously shown that a soluble form (sCADM1) generated by alternative splicing possesses a stop codon immediately downstream of the immunoglobulin-like domain. Here, we demonstrate the presence of sCADM1 in vivo and its possible role in neurite extension. sCADM1 appears to be a stromal protein because extracellular-restricted, but not intracellular-restricted, anti-CADM1 antibody stained stromal protein-rich extract from mouse brains. Murine plasmacytoma cells, P3U1, were modified to secrete sCADM1 fused with either immunoglobulin (Ig)G Fc portion (sCADM1-Fc) or its deletion form that lacks the immunoglobulin-like domain (ΔsCADM1-Fc). When P3U1 derivatives expressing sCADM1-Fc or ΔsCADM1-Fc were implanted into collagen gels, Fc-fused proteins were present more abundantly around the cells. Superior cervical ganglion neurons, parental P3U1, and either derivative were implanted into collagen gels separately, and co-cultured for 4 days. Bodian staining of the gel sections revealed that most superior cervical ganglion neurites turned toward the source of sCADM1-Fc, but not ΔsCADM1-Fc. Furthermore, immunofluorescence signals for sCADM1-Fc and membrane-bound CADM1 were co-localized on the neurite surface. These results show that sCADM1 appears to be involved in directional neurite extension by serving as an anchor to which membrane-bound CADM1 on the neurites can bind. PMID:19435791

  6. ACAP3 regulates neurite outgrowth through its GAP activity specific to Arf6 in mouse hippocampal neurons.

    PubMed

    Miura, Yuki; Hongu, Tsunaki; Yamauchi, Yohei; Funakoshi, Yuji; Katagiri, Naohiro; Ohbayashi, Norihiko; Kanaho, Yasunori

    2016-09-01

    ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3 in HEK (human embryonic kidney)-293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3 in hippocampal neurons. In addition, knockdown and knockout of Arf6 in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild-type, GTP- or GDP-locked mutant Arf6. Thus cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3 in concert with a guanine-nucleotide-exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons. PMID:27330119

  7. Enhanced Neural Cell Adhesion and Neurite Outgrowth on Graphene-Based Biomimetic Substrates

    PubMed Central

    Lee, Jong Ho; Kang, Seok Hee; Hwang, Eun Young; Hwang, Yu-Shik; Lee, Mi Hee; Park, Jong-Chul

    2014-01-01

    Neural cell adhesion and neurite outgrowth were examined on graphene-based biomimetic substrates. The biocompatibility of carbon nanomaterials such as graphene and carbon nanotubes (CNTs), that is, single-walled and multiwalled CNTs, against pheochromocytoma-derived PC-12 neural cells was also evaluated by quantifying metabolic activity (with WST-8 assay), intracellular oxidative stress (with ROS assay), and membrane integrity (with LDH assay). Graphene films were grown by using chemical vapor deposition and were then coated onto glass coverslips by using the scooping method. Graphene sheets were patterned on SiO2/Si substrates by using photolithography and were then covered with serum for a neural cell culture. Both types of CNTs induced significant dose-dependent decreases in the viability of PC-12 cells, whereas graphene exerted adverse effects on the neural cells just at over 62.5 ppm. This result implies that graphene and CNTs, even though they were the same carbon-based nanomaterials, show differential influences on neural cells. Furthermore, graphene-coated or graphene-patterned substrates were shown to substantially enhance the adhesion and neurite outgrowth of PC-12 cells. These results suggest that graphene-based substrates as biomimetic cues have good biocompatibility as well as a unique surface property that can enhance the neural cells, which would open up enormous opportunities in neural regeneration and nanomedicine. PMID:24592382

  8. Foxp2 Regulates Gene Networks Implicated in Neurite Outgrowth in the Developing Brain

    PubMed Central

    Vernes, Sonja C.; Oliver, Peter L.; Spiteri, Elizabeth; Lockstone, Helen E.; Puliyadi, Rathi; Taylor, Jennifer M.; Ho, Joses; Mombereau, Cedric; Brewer, Ariel; Lowy, Ernesto; Nicod, Jérôme; Groszer, Matthias; Baban, Dilair; Sahgal, Natasha; Cazier, Jean-Baptiste; Ragoussis, Jiannis; Davies, Kay E.; Geschwind, Daniel H.; Fisher, Simon E.

    2011-01-01

    Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections. PMID:21765815

  9. Non-prenylatable, cytosolic Rac1 alters neurite outgrowth while retaining the ability to be activated.

    PubMed

    Reddy, Jairus M; Samuel, Filsy G; McConnell, Jordan A; Reddy, Cristina P; Beck, Brian W; Hynds, DiAnna L

    2015-03-01

    Rac1 is an important regulator of axon extension, cell migration and actin reorganization. Like all Rho guanine triphosphatases (GTPases), Rac1 is targeted to the membrane by the addition of a geranylgeranyl moiety, an action thought to result in Rac1 guanosine triphosphate (GTP) binding. However, the role that Rac1 localization plays in its activation (GTP loading) and subsequent activation of effectors is not completely clear. To address this, we developed a non-prenylatable emerald green fluorescent protein (EmGFP)-Rac1 fusion protein (EmGFP-Rac1(C189A)) and assessed how expressing this construct affected neurite outgrowth, Rac1 localization and activation in neuroblastoma cells. Expression of EmGFP-Rac1(C189A) increased localization to the cytosol and induced cell clustering while increasing neurite initiation. EmGFP-Rac1(C189A) expression also increased Rac1 activation in the cytosol, compared to cells expressing wild-type Rac1 (EmGFP-Rac1). These results suggest that activation of Rac1 may not require plasma membrane localization, potentially leading to differential activation of cytosolic signaling pathways that alter cell morphology. Understanding the consequences of differential localization and activation of Rho GTPases, including Rac1, could lead to new therapeutic targets for treating neurological disorders. PMID:25479592

  10. Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building.

    PubMed

    Takeda, S; Yamazaki, H; Seog, D H; Kanai, Y; Terada, S; Hirokawa, N

    2000-03-20

    Kinesin superfamily proteins (KIFs) comprise several dozen molecular motor proteins. The KIF3 heterotrimer complex is one of the most abundantly and ubiquitously expressed KIFs in mammalian cells. To unveil the functions of KIF3, microinjection of function-blocking monovalent antibodies against KIF3 into cultured superior cervical ganglion (SCG) neurons was carried out. They significantly blocked fast axonal transport and brought about inhibition of neurite extension. A yeast two-hybrid binding assay revealed the association of fodrin with the KIF3 motor through KAP3. This was further confirmed by using vesicles collected from large bundles of axons (cauda equina), from which membranous vesicles could be prepared in pure preparations. Both immunoprecipitation and immunoelectron microscopy indicated the colocalization of fodrin and KIF3 on the same vesicles, the results reinforcing the evidence that the cargo of the KIF3 motor consists of fodrin-associating vesicles. In addition, pulse-labeling study implied partial comigration of both molecules as fast flow components. Taken together, the KIF3 motor is engaged in fast axonal transport that conveys membranous components important for neurite extension. PMID:10725338

  11. A patterned recombinant human IgM guides neurite outgrowth of CNS neurons

    NASA Astrophysics Data System (ADS)

    Xu, Xiaohua; Wittenberg, Nathan J.; Jordan, Luke R.; Kumar, Shailabh; Watzlawik, Jens O.; Warrington, Arthur E.; Oh, Sang-Hyun; Rodriguez, Moses

    2013-07-01

    Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks.

  12. A patterned recombinant human IgM guides neurite outgrowth of CNS neurons

    PubMed Central

    Xu, Xiaohua; Wittenberg, Nathan J.; Jordan, Luke R.; Kumar, Shailabh; Watzlawik, Jens O.; Warrington, Arthur E.; Oh, Sang-Hyun; Rodriguez, Moses

    2013-01-01

    Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks. PMID:23881231

  13. Growth

    NASA Astrophysics Data System (ADS)

    Waag, Andreas

    This chapter is devoted to the growth of ZnO. It starts with various techniques to grow bulk samples and presents in some detail the growth of epitaxial layers by metal organic chemical vapor deposition (MOCVD), molecular beam epitaxy (MBE), and pulsed laser deposition (PLD). The last section is devoted to the growth of nanorods. Some properties of the resulting samples are also presented. If a comparison between GaN and ZnO is made, very often the huge variety of different growth techniques available to fabricate ZnO is said to be an advantage of this material system. Indeed, growth techniques range from low cost wet chemical growth at almost room temperature to high quality MOCVD growth at temperatures above 1, 000∘C. In most cases, there is a very strong tendency of c-axis oriented growth, with a much higher growth rate in c-direction as compared to other crystal directions. This often leads to columnar structures, even at relatively low temperatures. However, it is, in general, not straight forward to fabricate smooth ZnO thin films with flat surfaces. Another advantage of a potential ZnO technology is said to be the possibility to grow thin films homoepitaxially on ZnO substrates. ZnO substrates are mostly fabricated by vapor phase transport (VPT) or hydrothermal growth. These techniques are enabling high volume manufacturing at reasonable cost, at least in principle. The availability of homoepitaxial substrates should be beneficial to the development of ZnO technology and devices and is in contrast to the situation of GaN. However, even though a number of companies are developing ZnO substrates, only recently good quality substrates have been demonstrated. However, these substrates are not yet widely available. Still, the situation concerning ZnO substrates seems to be far from low-cost, high-volume production. The fabrication of dense, single crystal thin films is, in general, surprisingly difficult, even when ZnO is grown on a ZnO substrate. However

  14. Novel Roles and Mechanism for Krüppel-like Factor 16 (KLF16) Regulation of Neurite Outgrowth and Ephrin Receptor A5 (EphA5) Expression in Retinal Ganglion Cells.

    PubMed

    Wang, Jianbo; Galvao, Joana; Beach, Krista M; Luo, Weijia; Urrutia, Raul A; Goldberg, Jeffrey L; Otteson, Deborah C

    2016-08-26

    Regenerative medicine holds great promise for the treatment of degenerative retinal disorders. Krüppel-like factors (KLFs) are transcription factors that have recently emerged as key tools in regenerative medicine because some of them can function as epigenetic reprogrammers in stem cell biology. Here, we show that KLF16, one of the least understood members of this family, is a POU4F2 independent transcription factor in retinal ganglion cells (RGCs) as early as embryonic day 15. When overexpressed, KLF16 inhibits RGC neurite outgrowth and enhances RGC growth cone collapse in response to exogenous ephrinA5 ligands. Ephrin/EPH signaling regulates RGC connectivity. The EphA5 promoter contains multiple GC- and GT-rich KLF-binding sites, which, as shown by ChIP-assays, bind KLF16 in vivo In electrophoretic mobility shift assays, KLF16 binds specifically to a single KLF site near the EphA5 transcription start site that is required for KLF16 transactivation. Interestingly, methylation of only six of 98 CpG dinucleotides within the EphA5 promoter blocks its transactivation by KLF16 but enables transactivation by KLF2 and KLF15. These data demonstrate a role for KLF16 in regulation of RGC neurite outgrowth and as a methylation-sensitive transcriptional regulator of EphA5 expression. Together, these data identify differential low level methylation as a novel mechanism for regulating KLF16-mediated EphA5 expression across the retina. Because of the critical role of ephrin/EPH signaling in patterning RGC connectivity, understanding the role of KLFs in regulating neurite outgrowth and Eph receptor expression will be vital for successful restoration of functional vision through optic nerve regenerative therapies. PMID:27402841

  15. Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

    EPA Science Inventory

    Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

  16. Antioxidants have a rapid and long-lasting effect on neuritic abnormalities in APP:PS1 mice.

    PubMed

    Garcia-Alloza, Monica; Borrelli, Laura A; Hyman, Bradley T; Bacskai, Brian J

    2010-12-01

    Senile plaques are a major pathological hallmark of Alzheimer's disease (AD). Compelling evidence suggests that senile plaques lead to structural alterations of neuronal processes and that local toxicity may be mediated by increased oxidative stress. Anti-oxidant therapy can alleviate the neuronal abnormalities in APP mice, but the time-course of this beneficial effect is unknown. We used multiphoton microscopy to assess in vivo the characteristics of antioxidant treatment on senile plaques and neurites in AD model mice (APPswe/PS1dE9). We observed that α-phenyl-N-tert-butyl nitrone (PBN), Ginkgo biloba extract (EGb 761) and Trolox had no effect on the size of existing senile plaques. However, all anti-oxidants had a straightening effect on curved neurites. This effect was detected as soon as 4 days after commencing the treatment, and was maintained after 1 month of daily treatment, with no further increase in the effect. The straightening of neurites persisted 15 days after stopping the treatment. These data indicate that neuronal plasticity is fast and still active in adult animals, and suggest that amelioration of the neuritic distortions associated with senile plaques with antioxidants is both rapid and long lasting. PMID:19124175

  17. MAGNETIC FIELD INFLUENCE ON NGF-STIMULATED NEURITE OUTGROWTH IN PC-12 CELLS: EFFECT OF PAINT FUMES

    EPA Science Inventory

    MAGNETIC FIELD INFLUENCE ON NGF-STIMULATED NEURITE OUTGROWTH IN PC-12 CELLS: EFFECT OF PAINT FUMES. C. F. Blackman1, D. E. House2*, S. G. Benane3*, A. Ubeda4, M.A. TrilIo4. 1 National Health and Environmental Effects Research Laboratory, EPA,
    Research Triangle Park, North Caro...

  18. On-Chip Multichannel Action Potential Recording System for Electrical Measurement of Single Neurites of Neuronal Network

    NASA Astrophysics Data System (ADS)

    Suzuki, Ikurou; Hattori, Akihiro; Yasuda, Kenji

    2007-11-01

    We have developed a multielectrode array recording system for single-neurite-firing measurement using an artificially constructed neuronal network on a chip, which has a 10 μm diameter array with electrodes spaced at 50 μm, for noninvasive 64-channel 100 kHz multirecording and the stimulation of a plurality of neurites extending from a single neuron. To improve the signal/noise ratio, the ground plane was set on the multi-electrode-array plane and platinum black was set on each of the 10 μm electrodes. Using this system, we performed a multisite recording of neurites of a single neuron of a rat hippocampal network in cases of both spontaneous firing and evoked responses to electrical stimulations, and estimated the velocity of action potential propagation among neurites of a single neuron from six recording sites. This demonstrated the potential use of our low-noise chip and our high-speed measurement system for the analysis of neuronal network activities at the single-neuron level.

  19. Laminar stream of detergents for subcellular neurite damage in a microfluidic device: a simple tool for the study of neuroregeneration

    NASA Astrophysics Data System (ADS)

    Lee, Chang Young; Romanova, Elena V.; Sweedler, Jonathan V.

    2013-06-01

    Objective. The regeneration and repair of damaged neuronal networks is a difficult process to study in vivo, leading to the development of multiple in vitro models and techniques for studying nerve injury. Here we describe an approach for generating a well-defined subcellular neurite injury in a microfluidic device. Approach. A defined laminar stream of sodium dodecyl sulfate (SDS) was used to damage selected portions of neurites of individual neurons. The somata and neurites unaffected by the SDS stream remained viable, thereby enabling the study of neuronal regeneration. Main results. By using well-characterized neurons from Aplysia californica cultured in vitro, we demonstrate that our approach is useful in creating neurite damage, investigating neurotrophic factors, and monitoring somata migration during regeneration. Supplementing the culture medium with acetylcholinesterase (AChE) or Aplysia hemolymph facilitated the regeneration of the peptidergic Aplysia neurons within 72 h, with longer (p < 0.05) and more branched (p < 0.05) neurites than in the control medium. After the neurons were transected, their somata migrated; intriguingly, for the control cultures, the migration direction was always away from the injury site (7/7). In the supplemented cultures, the number decreased to 6/8 in AChE and 4/8 in hemolymph, with reduced migration distances in both cases. Significance. The SDS transection approach is simple and inexpensive, yet provides flexibility in studying neuroregeneration, particularly when it is important to make sure there are no retrograde signals from the distal segments affecting regeneration. Neurons are known to not only be under tension but also balanced in terms of force, and the balance is obviously disrupted by transection. Our experimental platform, verified with Aplysia, can be extended to mammalian systems, and help us gain insight into the role that neurotrophic factors and mechanical tension play during neuronal regeneration.

  20. Mannosylerythritol lipid increases levels of galactoceramide in and neurite outgrowth from PC12 pheochromocytoma cells.

    PubMed

    Shibahara, M; Zhao, X; Wakamatsu, Y; Nomura, N; Nakahara, T; Jin, C; Nagaso, H; Murata, T; Yokoyama, K K

    2000-07-01

    We report here that a microbial extracellular glycolipid,mannosylerythritol lipid (MEL), induces the outgrowth ofneurites from and enhances the activity of acetylcholinesterase(AChE) in PC12 pheochromocytoma cells. Furthermore, treatment ofPC12 cells with MEL increased levels of galactosylceramide(Galbeta1-1'Cer; GalCer). Exposure of PC12 cells to exogenous GalCer caused the dose-dependent outgrowth ofneurites. By contrast, treatment of PC12 cells with nerve growthfactor (NGF) did not increase the level of GalCer in the cells. The neurite-related morphological changes induced by GalCerdifferend from those induced by NGF, indicating differencesbetween the signal transduction pathways triggered by NGF and by GalCer. PMID:19002832

  1. Neurotransmitters and synaptic components in the Merkel cell-neurite complex, a gentle touch receptor

    PubMed Central

    Maksimovic, Srdjan; Baba, Yoshichika; Lumpkin, Ellen A.

    2013-01-01

    Merkel cells are an enigmatic group of rare cells found in the skin of vertebrates. Most make contacts with somatosensory afferents to form Merkel cell-neurite complexes, which are gentle-touch receptors that initiate slowly adapting type I responses. The function of Merkel cells within the complex remains debated despite decades of research. Numerous anatomical studies demonstrate that Merkel cells form synaptic-like contacts with sensory afferent terminals. Moreover, recent molecular analysis reveals that Merkel cells express dozens of presynaptic molecules that are essential for synaptic vesicle release in neurons. Merkel cells also produce a host of neuro-active substances that can act as fast excitatory neurotransmitters or neuromodulators. Here, we review the major neurotransmitters found in Merkel cells and discuss these findings in relation to the potential function of Merkel cells in touch reception. PMID:23530998

  2. The Adhesion and Neurite Outgrowth of Neurons on Poly(D-lysine)/Hyaluronan Multilayer Films.

    PubMed

    Shi, Haifei; Sheng, Guoping

    2016-06-01

    Poly(D-lysine)/hyaluronan (PDL/HA) films were prepared using layer-by-layer assembly technique and chemically cross-linked with a water soluble carbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS) through formation of amide bonds. Quartz crystal microbalance with dissipation (QCM-D) was used to follow the cross-linking reaction. Atomic force measurement, ellipsometry, and Fourier transform infrared (FTIR) spectroscopy were performed to study the chemical structure, topography, thickness and mechanical properties of the cross-linked films. QCM-D and Frictional force study were used to reveal the viscoelasticity of the films after cross-linking treatment. The stability of the films was studied via incubating the films in physiological environment. Finally, the neurons were used to evaluate the interaction between films and cells. The results indicated that the neurons were preferably proliferating and outgrowth neurite on cross-linked films while uncross-linked films are highly cell resistant. PMID:27427590

  3. Centlein, a novel microtubule-associated protein stabilizing microtubules and involved in neurite formation.

    PubMed

    Jing, Zhenli; Yin, Huilong; Wang, Pan; Gao, Juntao; Yuan, Li

    2016-04-01

    We have previously reported that the centriolar protein centlein functions as a molecular link between C-Nap1 and Cep68 to maintain centrosome cohesion [1]. In this study, we identified centlein as a novel microtubule-associated protein (MAP), directly binding to purified microtubules (MTs) via its longest coiled-coil domain. Overexpression of centlein caused profound nocodazole- and cold-resistant MT bundles, which also relied on its MT-binding domain. siRNA-mediated centlein depletion resulted in a significant reduction in tubulin acetylation level and overall fluorescence intensity of cytoplasmic MT acetylation. Centlein was further characterized in neurons. We found that centlein overexpression inhibited neurite formation in retinoic acid (RA)-induced SH-SY5Y and N2a cells. Taken together, we propose that centlein is involved in MT stability and neuritogenesis in vivo. PMID:26915804

  4. Whole-exome sequencing and neurite outgrowth analysis in autism spectrum disorder.

    PubMed

    Hashimoto, Ryota; Nakazawa, Takanobu; Tsurusaki, Yoshinori; Yasuda, Yuka; Nagayasu, Kazuki; Matsumura, Kensuke; Kawashima, Hitoshi; Yamamori, Hidenaga; Fujimoto, Michiko; Ohi, Kazutaka; Umeda-Yano, Satomi; Fukunaga, Masaki; Fujino, Haruo; Kasai, Atsushi; Hayata-Takano, Atsuko; Shintani, Norihito; Takeda, Masatoshi; Matsumoto, Naomichi; Hashimoto, Hitoshi

    2016-03-01

    Autism spectrum disorder (ASD) is a complex group of clinically heterogeneous neurodevelopmental disorders with unclear etiology and pathogenesis. Genetic studies have identified numerous candidate genetic variants, including de novo mutated ASD-associated genes; however, the function of these de novo mutated genes remains unclear despite extensive bioinformatics resources. Accordingly, it is not easy to assign priorities to numerous candidate ASD-associated genes for further biological analysis. Here we developed a convenient system for identifying an experimental evidence-based annotation of candidate ASD-associated genes. We performed trio-based whole-exome sequencing in 30 sporadic cases of ASD and identified 37 genes with de novo single-nucleotide variations (SNVs). Among them, 5 of those 37 genes, POGZ, PLEKHA4, PCNX, PRKD2 and HERC1, have been previously reported as genes with de novo SNVs in ASD; and consultation with in silico databases showed that only HERC1 might be involved in neural function. To examine whether the identified gene products are involved in neural functions, we performed small hairpin RNA-based assays using neuroblastoma cell lines to assess neurite development. Knockdown of 8 out of the 14 examined genes significantly decreased neurite development (P<0.05, one-way analysis of variance), which was significantly higher than the number expected from gene ontology databases (P=0.010, Fisher's exact test). Our screening system may be valuable for identifying the neural functions of candidate ASD-associated genes for further analysis and a substantial portion of these genes with de novo SNVs might have roles in neuronal systems, although further detailed analysis might eliminate false positive genes from identified candidate ASD genes. PMID:26582266

  5. Whole-exome sequencing and neurite outgrowth analysis in autism spectrum disorder

    PubMed Central

    Hashimoto, Ryota; Nakazawa, Takanobu; Tsurusaki, Yoshinori; Yasuda, Yuka; Nagayasu, Kazuki; Matsumura, Kensuke; Kawashima, Hitoshi; Yamamori, Hidenaga; Fujimoto, Michiko; Ohi, Kazutaka; Umeda-Yano, Satomi; Fukunaga, Masaki; Fujino, Haruo; Kasai, Atsushi; Hayata-Takano, Atsuko; Shintani, Norihito; Takeda, Masatoshi; Matsumoto, Naomichi; Hashimoto, Hitoshi

    2016-01-01

    Autism spectrum disorder (ASD) is a complex group of clinically heterogeneous neurodevelopmental disorders with unclear etiology and pathogenesis. Genetic studies have identified numerous candidate genetic variants, including de novo mutated ASD-associated genes; however, the function of these de novo mutated genes remains unclear despite extensive bioinformatics resources. Accordingly, it is not easy to assign priorities to numerous candidate ASD-associated genes for further biological analysis. Here we developed a convenient system for identifying an experimental evidence-based annotation of candidate ASD-associated genes. We performed trio-based whole-exome sequencing in 30 sporadic cases of ASD and identified 37 genes with de novo single-nucleotide variations (SNVs). Among them, 5 of those 37 genes, POGZ, PLEKHA4, PCNX, PRKD2 and HERC1, have been previously reported as genes with de novo SNVs in ASD; and consultation with in silico databases showed that only HERC1 might be involved in neural function. To examine whether the identified gene products are involved in neural functions, we performed small hairpin RNA-based assays using neuroblastoma cell lines to assess neurite development. Knockdown of 8 out of the 14 examined genes significantly decreased neurite development (P<0.05, one-way analysis of variance), which was significantly higher than the number expected from gene ontology databases (P=0.010, Fisher's exact test). Our screening system may be valuable for identifying the neural functions of candidate ASD-associated genes for further analysis and a substantial portion of these genes with de novo SNVs might have roles in neuronal systems, although further detailed analysis might eliminate false positive genes from identified candidate ASD genes. PMID:26582266

  6. Carbon disulfide inhibits neurite outgrowth and neuronal migration of dorsal root ganglion in vitro.

    PubMed

    Ding, Ning; Xiang, Yujuan; Jiang, Hao; Zhang, Weiwei; Liu, Huaxiang; Li, Zhenzhong

    2011-12-01

    Carbon disulfide (CS₂) is a neurotoxic industrial solvent and widely used in the vulcanization of rubber, rayon, cellophane, and adhesives. Although the neurotoxicity of CS₂ has been recognized for over a century, the precise mechanism of neurotoxic action of CS₂ remains unknown. In the present study, a embryonic rat dorsal root ganglia (DRG) explants culture model was established. Using the organotypic DRG cultures, the direct neurotoxic effects of CS₂ on outgrowth of neurites and migration of neurons from DRG explants were investigated. The organotypic DRG cultures were exposed to different concentrations of CS₂ (0.01 mmol/L, 0.1 mmol/L, 1 mmol/L). The number of nerve fiber bundles extended from DRG explants decreased significantly in the presence of CS₂ (0.01 mmol/L, 15.00 ± 2.61, p < .05; 0.1 mmol/L, 11.17 ± 1.47, p < .001; 1 mmol/L, 8.00 ± 1.41, p < .001) as compared with that in the absence of CS₂ (17.83 ± 2.48). The number of neurons migrated from DRG explants decreased significantly in the presence of CS₂ (0.01 mmol/L, 79.50 ± 9.40, p < .01; 0.1 mmol/L, 62.50 ± 14.15, p < .001; 1 mmol/L, 34.67 ± 7.58, p < .001) as compared with that in the absence of CS₂ (99.33 ± 15.16). And also, the decreases in the number of nerve fiber bundles and migrated DRG neurons were in a dose-dependent manner of CS₂. These data implicated that CS₂ could inhibit neurite outgrowth and neuronal migration from DRG explants in vitro. PMID:21777162

  7. FGF-2 deficiency causes dysregulation of Arhgef6 and downstream targets in the cerebral cortex accompanied by altered neurite outgrowth and dendritic spine morphology.

    PubMed

    Baum, Philip; Vogt, Miriam A; Gass, Peter; Unsicker, Klaus; von Bohlen Und Halbach, Oliver

    2016-05-01

    Fibroblast growth factor 2 (FGF-2) is an abundant growth factor in the brain and exerts multiple functions on neural cells ranging from cell division, cell fate determination to differentiation. However, many details of the molecular mechanisms underlying the diverse functions of FGF-2 are poorly understood. In a comparative microarray analysis of motor sensory cortex (MSC) tissue of adult knockout (FGF-2(-/-)) and control (FGF-2(+/+)) mice, we found a substantial number of regulated genes, which are implicated in cytoskeletal machinery dynamics. Specifically, we found a prominent downregulation of Arhgef6. Arhgef6 mRNA was significantly reduced in the FGF-2(-/-) cortex, and Arhgef6 protein virtually absent, while RhoA protein levels were massively increased and Cdc42 protein levels were reduced. Since Arhgef6 is localized to dendritic spines, we next analyzed dendritic spines of adult FGF2(-/-) and control mouse cortices. Spine densities were significantly increased, whereas mean length of spines on dendrites of layer V of MSC neurons in adult FGF-2(-/-) mice was significantly decreased as compared to respective controls. Furthermore, neurite length in dissociated cortical cultures from E18 FGF-2(-/-) mice was significantly reduced at DIV7 as compared to wildtype neurons. Despite the fact that altered neuronal morphology and alterations in dendritic spines were observed, FGF-2(-/-) mice behave relatively unsuspicious in several behavioral tasks. However, FGF-2(-/-) mice exhibited decreased thermal pain sensitivity in the hotplate-test. PMID:26970009

  8. Quantitative assessment of neurite outgrowth in human embryonic stem cell derived hN2 cells using automated high-content image analysis

    EPA Science Inventory

    Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary ne...

  9. Division of labor in the growth cone by DSCR1.

    PubMed

    Catlett, Timothy S; Gomez, Timothy M

    2016-05-23

    Local protein synthesis directs growth cone turning of nascent axons, but mechanisms governing this process within compact, largely autonomous microenvironments remain poorly understood. In this issue, Wang et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201510107) demonstrate that the calcineurin regulator Down syndrome critical region 1 protein modulates both basal neurite outgrowth and growth cone turning. PMID:27216257

  10. Characterization of the role of full-length CRMP3 and its calpain-cleaved product in inhibiting microtubule polymerization and neurite outgrowth

    SciTech Connect

    Aylsworth, Amy; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario ; Jiang, Susan X.; Desbois, Angele; Hou, Sheng T.

    2009-10-01

    Collapsin response mediator proteins (CRMPs) are key modulators of cytoskeletons during neurite outgrowth in response to chemorepulsive guidance molecules. However, their roles in adult injured neurons are not well understood. We previously demonstrated that CRMP3 underwent calcium-dependent N-terminal protein cleavage during excitotoxicity-induced neurite retraction and neuronal death. Here, we report findings that the full-length CRMP3 inhibits tubulin polymerization and neurite outgrowth in cultured mature cerebellar granule neurons, while the N-terminal truncated CRMP3 underwent nuclear translocation and caused a significant nuclear condensation. The N-terminal truncated CRMP3 underwent nuclear translocation through nuclear pores. Nuclear protein pull-down assay and mass spectrometry analysis showed that the N-terminal truncated CRMP3 was associated with nuclear vimentin. In fact, nuclear-localized CRMP3 co-localized with vimentin during glutamate-induced excitotoxicity. However, the association between the truncated CRMP3 and vimentin was not critical for nuclear condensation and neurite outgrowth since over-expression of truncated CRMP3 in vimentin null neurons did not alleviate nuclear condensation and neurite outgrowth inhibition. Together, these studies showed CRMP3's role in attenuating neurite outgrowth possibility through inhibiting microtubule polymerization, and also revealed its novel association with vimentin during nuclear condensation prior to neuronal death.

  11. The Roles of Genes in the Neuronal Migration and Neurite Outgrowth Network in Developmental Dyslexia: Single- and Multiple-Risk Genetic Variants.

    PubMed

    Shao, Shanshan; Kong, Rui; Zou, Li; Zhong, Rong; Lou, Jiao; Zhou, Jie; Guo, Shengnan; Wang, Jia; Zhang, Xiaohui; Zhang, Jiajia; Song, Ranran

    2016-08-01

    Abnormal regulation of neural migration and neurite growth is thought to be an important feature of developmental dyslexia (DD). We investigated 16 genetic variants, selected by bioinformatics analyses, in six key genes in the neuronal migration and neurite outgrowth network in a Chinese population. We first observed that KIAA0319L rs28366021, KIAA0319 rs4504469, and DOCK4 rs2074130 were significantly associated with DD risk after false discovery rate (FDR) adjustment for multiple comparisons (odds ratio (OR) = 0.672, 95 % confidence interval (CI) = 0.505-0.894, P = 0.006; OR = 1.608, 95 % CI = 1.174-2.203, P = 0.003; OR = 1.681, 95 % CI = 1.203-2.348, P = 0.002). The following classification and regression tree (CART) analysis revealed a prediction value of gene-gene interactions among DOCK4 rs2074130, KIAA0319 rs4504469, DCDC2 rs2274305, and KIAA0319L rs28366021 variants. Compared with the lowest risk carriers of the combination of rs2074130 CC, rs4504469 CC, and rs2274305 GG genotype, individuals carrying the combined genotypes of rs2074130 CC, rs4504469 CT or TT, and rs28366021 GG had a significantly increased risk for DD (OR = 2.492, 95 % CI = 1.447-4.290, P = 0.001); individuals with the combination of rs2074130 CT or TT and rs28366021 GG genotype exhibited the highest risk for DD (OR = 2.770, 95 % CI = 2.265-6.276, P = 0.000). A significant dose effect was observed among these four variants (P for trend = 0.000). In summary, this study supports the importance of single- and multiple-risk variants in this network in DD susceptibility in China. PMID:26184631

  12. NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition

    PubMed Central

    Caltharp, Shelley A; Pira, Charmaine U; Mishima, Noboru; Youngdale, Erik N; McNeill, David S; Liwnicz, Boleslaw H; Oberg, Kerby C

    2007-01-01

    Background Nogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS) and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition. Results We isolated chicken NOGO-A and determined its sequence. A multiple alignment of the amino acid sequence across divergent species, identified five previously undescribed, Nogo-A specific conserved regions that may be relevant for development. NOGO gene transcripts (NOGO-A, NOGO-B and NOGO-C) were differentially expressed in the CNS during development and a second NOGO-A splice variant was identified. We further localized NOGO-A expression during key phases of CNS development by in situ hybridization. CNS-associated NOGO-A was induced coincident with neural plate formation and up-regulated by FGF in the transformation of non-neural ectoderm into neural precursors. NOGO-A expression was diffuse in the neuroectoderm during the early proliferative phase of development, and migration, but localized to large projection neurons of the optic tectum and tectal-associated nuclei during architectural differentiation, lamination and network establishment. Conclusion These data suggest Nogo-A plays a functional role in the determination of neural identity and/or differentiation and also appears to play a later role in the networking of large projection neurons during neurite formation and synaptogenesis. These data indicate that Nogo-A is a multifunctional protein with additional roles during CNS development that are disparate from its later role of neurite outgrowth inhibition in the adult CNS. PMID:17433109

  13. The importance of EHD1 in neurite outgrowth contributing to the functional recovery after spinal cord injury.

    PubMed

    Wu, Chunshuai; Cui, Zhiming; Liu, Yonghua; Zhang, Jinlong; Ding, Wensen; Wang, Song; Bao, Guofeng; Xu, Guanhua; Sun, Yuyu; Chen, Jiajia

    2016-08-01

    Traumatic spinal cord injury is one of the most common and severe problems for using NGF to promote the neurite outgrowth of survival neurons. EHD1 regulates and controls the endocytosis and transportation of neurotrophins and transmembrane cargo via recycling endosome for neurite outgrowth. TrkA is particularly considered to be a functional specific recepter in the cell membrane for NGF and is activated upon NGF binding. The transcytosis of TrkA is dependent on Rab11 recycling endosomes and is promoted by NGF signaling itself at the axon terminal. In this study, we established an acute spinal cord contusion injury model in adult rats to investigate the potential role of EHD1 during the pathological process of SCI. Western blot analysis suggested that EHD1 expression was low in the sham-operated adult rat spinal cords and was significantly up-regulated 1d after injury. Immunohistochemical staining detected the general distribution of EHD1 protein in both the gray and white matter of adult rat spinal cords. Double immunofluorescent staining indicated that EHD1 was expressed in neurons, astrocytes and microglias in the adult rat spinal cord, and obvious changes of EHD1 expression occurred in neurons during SCI pathological process. Significant up-regulation of EHD1 expression was observed in MAP2 positive neurons at 1 day after SCI, in comparison with the sham-operated control, which indicated that EHD1 might play a vital role in neurite outgrowth. Our data indicated that EHD1 could interact with TrkA, and is in the upstream of TrkA. EHD1 up-regulated the expression of TrkA in the glutamate stimulated primary neurons. Based on our experimental data, we boldly conclude that EHD1 regulates the recycling of TrkA back to cell membrane, improving the utilization efficiency of the NGF, which is vital for neurite outgrowth and functional recovery after spinal cord injury. PMID:27211346

  14. Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1

    PubMed Central

    Ma, Qi; Hu, Qing-song; Xu, Ran-jie; Zhen, Xue-chu; Wang, Guang-hui

    2015-01-01

    Aim: Omi is an ATP-independent serine protease that is necessary for neuronal function and survival. The aim of this study was to investigate the role of protease Omi in regulating differentiation of mouse neuroblastoma cells and to identify the substrate of Omi involved in this process. Methods: Mouse neuroblastoma N2a cells and Omi protease-deficient mnd2 mice were used in this study. To modulate Omi and E2F1 expression, N2a cells were transfected with expression plasmids, shRNA plasmids or siRNA. Protein levels were detected using immunoblot assays. The interaction between Omi and E2F1 was studied using immunoprecipitation, GST pulldown and in vitro cleavage assays. N2a cells were treated with 20 μmol/L retinoic acid (RA) and 1% fetal bovine serum to induce neurite outgrowth, which was measured using Image J software. Results: E2F1 was significantly increased in Omi knockdown cells and in brain lysates of mnd2 mice, and was decreased in cells overexpressing wild-type Omi, but not inactive Omi S276C. In brain lysates of mnd2 mice, endogenous E2F1 was co-immunoprecipitated with endogenous Omi. In vitro cleavage assay demonstrated that Omi directly cleaved E2F1. Treatment of N2a cells with RA induced marked differentiation and neurite outgrowth accompanied by significantly increased Omi and decreased E2F1 levels, which were suppressed by pretreatment with the specific Omi inhibitor UCF-101. Knockdown of Omi in N2a cells suppressed RA-induced neurite outgrowth, which was partially restored by knockdown of E2F1. Conclusion: Protease Omi facilitates neurite outgrowth by cleaving the transcription factor E2F1 in differentiated neuroblastoma cells; E2F1 is a substrate of Omi. PMID:26238290

  15. Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo.

    PubMed

    Jørgensen, Jesper Roland; Fransson, Anette; Fjord-Larsen, Lone; Thompson, Lachlan H; Houchins, Jeffrey P; Andrade, Nuno; Torp, Malene; Kalkkinen, Nisse; Andersson, Elisabet; Lindvall, Olle; Ulfendahl, Mats; Brunak, Søren; Johansen, Teit E; Wahlberg, Lars U

    2012-01-01

    Neurotrophic factors are secreted proteins responsible for migration, growth and survival of neurons during development, and for maintenance and plasticity of adult neurons. Here we present a novel secreted protein named Cometin which together with Meteorin defines a new evolutionary conserved protein family. During early mouse development, Cometin is found exclusively in the floor plate and from E13.5 also in dorsal root ganglions and inner ear but apparently not in the adult nervous system. In vitro, Cometin promotes neurite outgrowth from dorsal root ganglion cells which can be blocked by inhibition of the Janus or MEK kinases. In this assay, additive effects of Cometin and Meteorin are observed indicating separate receptors. Furthermore, Cometin supports migration of neuroblasts from subventricular zone explants to the same extend as stromal cell derived factor 1a. Given the neurotrophic properties in vitro, combined with the restricted inner ear expression during development, we further investigated Cometin in relation to deafness. In neomycin deafened guinea pigs, two weeks intracochlear infusion of recombinant Cometin supports spiral ganglion neuron survival and function. In contrast to the control group receiving artificial perilymph, Cometin treated animals retain normal electrically-evoked brainstem response which is maintained several weeks after treatment cessation. Neuroprotection is also evident from stereological analysis of the spiral ganglion. Altogether, these studies show that Cometin is a potent new neurotrophic factor with therapeutic potential. PMID:21985865

  16. Proteomic analysis of differentiating neuroblastoma cells treated with sub-lethal neurite inhibitory concentrations of diazinon: Identification of novel biomarkers of effect

    SciTech Connect

    Harris, W.; Sachana, M.; Flaskos, J.; Hargreaves, A.J.

    2009-10-15

    In previous work we showed that sub-lethal levels of diazinon inhibited neurite outgrowth in differentiating N2a neuroblastoma cells. Western blotting analysis targeted at proteins involved in axon growth and stress responses, revealed that such exposure led to a reduction in the levels of neurofilament heavy chain, microtubule associated protein 1 B (MAP 1B) and HSP-70. The aim of this study was to apply the approach of 2 dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify novel biomarkers of effect. A number of proteins were found to be up-regulated compared to the control on silver-stained gels. These were classified in to 3 main groups of proteins: cytosolic factors, chaperones and the actin-binding protein cofilin, all of which are involved in cell differentiation, survival or metabolism. The changes observed for cofilin were further confirmed by quantitative Western blotting analysis with anti-actin and anti-cofilin antibodies. Indirect immunofluorescence staining with the same antibodies indicated that the microfilament network was disrupted in diazinon-treated cells. Our data suggest that microfilament organisation is disrupted by diazinon exposure, which may be related to increased cofilin expression.

  17. Glypican-2 binds to midkine: the role of glypican-2 in neuronal cell adhesion and neurite outgrowth.

    PubMed

    Kurosawa, N; Chen, G Y; Kadomatsu, K; Ikematsu, S; Sakuma, S; Muramatsu, T

    2001-06-01

    Cell-surface heparan sulfate proteoglycans participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study was performed to elucidate whether glypican-2 plays a role in interactions of neurons with midkine (MK), a heparin-binding neuroregulatory factor. MK bound to heparan sulfate chains of glypican-2 in a manner similar to syndecan-3. Microbeads coated with MK or poly-L-lysine induced clustering of glypican-2 as well as syndecan-3. Substratum-bound MK or poly-L-lysine induced cell adhesion of N2a neuroblastoma cells, while only MK promoted neurite outgrowth of these cells. Ligation of cell-surface glypican-2 with MK or an antibody against epitope-tagged glypican-2 induced cell adhesion and promoted neurite outgrowth. These results verified that cell-surface glypican-2 bound to MK and suggested that MK-glypican-2 interactions participate in neuronal cell migration and neurite outgrowth. In addition, we observed different localization of epitope-tagged glypican-2 and syndecan-3 on the surface of N2a cells; the result suggested that they may play different roles in MK-mediated neural function. PMID:12084985

  18. Automated imaging system for fast quantitation of neurons, cell morphology and neurite morphometry in vivo and in vitro.

    PubMed

    Tapias, Victor; Greenamyre, J Timothy; Watkins, Simon C

    2013-06-01

    Quantitation of neurons using stereologic approaches reduces bias and systematic error, but is time-consuming and labor-intensive. Accurate methods for quantifying neurons in vitro are lacking; conventional methodologies are limited in reliability and application. The morphological properties of the soma and neurites are a key aspect of neuronal phenotype and function, but the assays commonly used in such evaluations are beset with several methodological drawbacks. Herein we describe automated techniques to quantify the number and morphology of neurons (or any cell type, e.g., astrocytes) and their processes with high speed and accuracy. Neuronal quantification from brain tissue using a motorized stage system yielded results that were statistically comparable to those generated by stereology. The approach was then adapted for in vitro neuron and neurite outgrowth quantification. To determine the utility of our methods, rotenone was used as a neurotoxicant leading to morphological changes in neurons and cell death, astrocytic activation, and loss of neurites. Importantly, our technique counted about 8 times as many neurons in less than 5-10% of the time taken by manual stereological analysis. PMID:23220621

  19. Non-obese diabetic mice rapidly develop dramatic sympathetic neuritic dystrophy: a new experimental model of diabetic autonomic neuropathy.

    PubMed

    Schmidt, Robert E; Dorsey, Denise A; Beaudet, Lucie N; Frederick, Kathy E; Parvin, Curtis A; Plurad, Santiago B; Levisetti, Matteo G

    2003-11-01

    To address the pathogenesis of diabetic autonomic neuropathy, we have examined the sympathetic nervous system in non-obese diabetic (NOD) and streptozotocin (STZ)-induced diabetic mice, two models of type 1 diabetes, and the db/db mouse, a model of type 2 diabetes. After only 3 to 5 weeks of diabetes, NOD mice developed markedly swollen axons and dendrites ("neuritic dystrophy") in the prevertebral superior mesenteric and celiac ganglia (SMG-CG), similar to the pathology described in diabetic STZ- and BBW-rat and man. Comparable changes failed to develop in the superior cervical ganglia of the NOD mouse or in the SMG-CG of non-diabetic NOD siblings. STZ-induced diabetic mice develop identical changes, although at a much slower pace and to a lesser degree than NOD mice. NOD-SCID mice, which are genetically identical to NOD mice except for the absence of T and B cells, do not develop diabetes or neuropathology comparable to diabetic NOD mice. However, STZ-treated NOD-SCID mice develop severe neuritic dystrophy, evidence against an exclusively autoimmune pathogenesis for autonomic neuropathy in this model. Chronically diabetic type 2 db/db mice fail to develop neuritic dystrophy, suggesting that hyperglycemia alone may not be the critical and sufficient element. The NOD mouse appears to be a valuable model of diabetic sympathetic autonomic neuropathy with unambiguous, rapidly developing neuropathology which corresponds closely to the characteristic pathology of other rodent models and man. PMID:14578206

  20. Automated imaging system for fast quantitation of neurons, cell morphology and neurite morphometry in vivo and in vitro

    PubMed Central

    Tapias, Victor; Greenamyre, J. Timothy; Watkins, Simon C.

    2012-01-01

    Quantitation of neurons using stereologic approaches reduces bias and systematic error, but is time-consuming and labor-intensive. Accurate methods for quantifying neurons in vitro are lacking; conventional methodologies are limited in reliability and application. The morphological properties of the soma and neurites are a key aspect of neuronal phenotype and function, but the assays commonly used in such evaluations are beset with several methodological drawbacks. Herein we describe automated techniques to quantify the number and morphology of neurons (or any cell type, e.g., astrocytes) and their processes with high speed and accuracy. Neuronal quantification from brain tissue using a motorized stage system yielded results that were statistically comparable to those generated by stereology. The approach was then adapted for in vitro neuron and neurite outgrowth quantification. To determine the utility of our methods, rotenone was used as a neurotoxicant leading to morphological changes in neurons and cell death, astrocytic activation, and loss of neurites. Importantly, our technique counted about 8 times as many neurons in less than 5–10% of the time taken by manual stereological analysis. PMID:23220621

  1. Lysophospholipid receptors LPA1–3 are not required for the inhibitory effects of LPA on mouse retinal growth cones

    PubMed Central

    Birgbauer, Eric; Chun, Jerold

    2016-01-01

    One of the major requirements in the development of the visual system is axonal guidance of retinal ganglion cells toward correct targets in the brain. A novel class of extracellular lipid signaling molecules, lysophospholipids, may serve as potential axon guidance cues. They signal through cognate G protein-coupled receptors, at least some of which are expressed in the visual system. Here we show that in the mouse visual system, a lysophospholipid known as lysophosphatidic acid (LPA) is inhibitory to retinal neurites in vitro when delivered extracellularly, causing growth cone collapse and neurite retraction. This inhibitory effect of LPA is both active in the nanomolar range and specific compared to the related lysophospholipid, sphingosine 1-phosphate (S1P). Knockout mice lacking three of the five known LPA receptors, LPA1–3, continue to display retinal growth cone collapse and neurite retraction in response to LPA, demonstrating that these three receptors are not required for these inhibitory effects and indicating the existence of one or more functional LPA receptors expressed on mouse retinal neurites that can mediate neurite retraction. PMID:26966392

  2. Centrosome movements in vivo correlate with specific neurite formation downstream of LIM homeodomain transcription factor activity.

    PubMed

    Andersen, Erica F; Halloran, Mary C

    2012-10-01

    Neurons must develop complex structure to form proper connections in the nervous system. The initiation of axons in defined locations on the cell body and their extension to synaptic targets are critical steps in neuronal morphogenesis, yet the mechanisms controlling axon formation in vivo are poorly understood. The centrosome has been implicated in multiple aspects of neuronal morphogenesis; however, its function in axon development is under debate. Conflicting results from studies of centrosome function in axonogenesis suggest that its role is context dependent and underscore the importance of studying centrosome function as neurons develop in their natural environment. Using live imaging of zebrafish Rohon-Beard (RB) sensory neurons in vivo, we discovered a spatiotemporal relationship between centrosome position and the formation of RB peripheral, but not central, axons. We tested centrosome function by laser ablation and found that centrosome disruption inhibited peripheral axon outgrowth. In addition, we show that centrosome position and motility are regulated by LIM homeodomain transcription factor activity, which is specifically required for the development of RB peripheral axons. Furthermore, we show a correlation between centrosome mislocalization and ectopic axon formation in bashful (laminin alpha 1) mutants. Thus, both intrinsic transcription factor activity and extracellular cues can influence centrosome position and axon formation in vivo. This study presents the first positive association between the centrosome and axon formation in vivo and suggests that the centrosome is important for differential neurite formation in neurons with complex axonal morphologies. PMID:22899847

  3. Novel High Content Screen Detects Compounds That Promote Neurite Regeneration from Cochlear Spiral Ganglion Neurons.

    PubMed

    Whitlon, Donna S; Grover, Mary; Dunne, Sara F; Richter, Sonja; Luan, Chi-Hao; Richter, Claus-Peter

    2015-01-01

    The bipolar spiral ganglion neurons (SGN) carry sound information from cochlear hair cells to the brain. After noise, antibiotic or toxic insult to the cochlea, damage to SGN and/or hair cells causes hearing impairment. Damage ranges from fiber and synapse degeneration to dysfunction and loss of cells. New interventions to regenerate peripheral nerve fibers could help reestablish transfer of auditory information from surviving or regenerated hair cells or improve results from cochlear implants, but the biochemical mechanisms to target are largely unknown. Presently, no drugs exist that are FDA approved to stimulate the regeneration of SGN nerve fibers. We designed an original phenotypic assay to screen 440 compounds of the NIH Clinical Collection directly on dissociated mouse spiral ganglia. The assay detected one compound, cerivastatin, that increased the length of regenerating neurites. The effect, mimicked by other statins at different optimal concentrations, was blocked by geranylgeraniol. These results demonstrate the utility of screening small compound libraries on mixed cultures of dissociated primary ganglia. The success of this screen narrows down a moderately sized library to a single compound which can be elevated to in-depth in vivo studies, and highlights a potential new molecular pathway for targeting of hearing loss drugs. PMID:26521685

  4. Novel High Content Screen Detects Compounds That Promote Neurite Regeneration from Cochlear Spiral Ganglion Neurons

    PubMed Central

    Whitlon, Donna S.; Grover, Mary; Dunne, Sara F.; Richter, Sonja; Luan, Chi-Hao; Richter, Claus-Peter

    2015-01-01

    The bipolar spiral ganglion neurons (SGN) carry sound information from cochlear hair cells to the brain. After noise, antibiotic or toxic insult to the cochlea, damage to SGN and/or hair cells causes hearing impairment. Damage ranges from fiber and synapse degeneration to dysfunction and loss of cells. New interventions to regenerate peripheral nerve fibers could help reestablish transfer of auditory information from surviving or regenerated hair cells or improve results from cochlear implants, but the biochemical mechanisms to target are largely unknown. Presently, no drugs exist that are FDA approved to stimulate the regeneration of SGN nerve fibers. We designed an original phenotypic assay to screen 440 compounds of the NIH Clinical Collection directly on dissociated mouse spiral ganglia. The assay detected one compound, cerivastatin, that increased the length of regenerating neurites. The effect, mimicked by other statins at different optimal concentrations, was blocked by geranylgeraniol. These results demonstrate the utility of screening small compound libraries on mixed cultures of dissociated primary ganglia. The success of this screen narrows down a moderately sized library to a single compound which can be elevated to in-depth in vivo studies, and highlights a potential new molecular pathway for targeting of hearing loss drugs. PMID:26521685

  5. Neuritic and Diffuse Plaque Associations with Memory in Non-Cognitively Impaired Elderly.

    PubMed

    Malek-Ahmadi, Michael; Perez, Sylvia E; Chen, Kewei; Mufson, Elliott J

    2016-07-14

    The presence of Alzheimer's disease (AD)-related neuropathology among cognitively normal individuals has been well documented. It has been proposed that these individuals may represent a pre-clinical AD population. Previous studies have demonstrated a negative association between the presence of both amyloid-β (Aβ) plaques and neurofibrillary tangles with ante-mortem cognitive performance, a relationship which is likely influenced by a number of factors including age and APOE ɛ4 carrier status. The present study determined whether the presence of neuritic plaques (NPs) and diffuse plaques (DPs) are associated with performance in a number of cognitive domains after accounting for APOE ɛ4 carrier status and neurofibrillary tangle presence in a cohort of 123 older participants from the Rush Religious Order Study who died with a premortem clinical diagnosis of no cognitive impairment (NCI). After adjusting for age at death, education, gender, Braak stage, and APOE ɛ4 carrier status, the presence of NPs was associated with lower performance in the cognitive domains of Global Cognition (p = 0.002), Episodic Memory (p = 0.03), Semantic Memory (p = 0.009), and Visuospatial performance (p = 0.006), while DPs showed no association with any cognitive domain examined. These results suggest that decreases in cognition in elderly NCI individuals are associated with an increase in NPs and not DPs when age at death, education, gender, APOE ɛ4 status, and Braak stage are taken into consideration. PMID:27540968

  6. Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans.

    PubMed

    Zheng, Chaogu; Diaz-Cuadros, Margarete; Chalfie, Martin

    2015-10-27

    Wnt proteins regulate axonal outgrowth along the anterior-posterior axis, but the intracellular mechanisms that modulate the strength of Wnt signaling in axon guidance are largely unknown. Using the Caenorhabditis elegans mechanosensory PLM neurons, we found that posteriorly enriched LIN-44/Wnt acts as a repellent to promote anteriorly directed neurite outgrowth through the LIN-17/Frizzled receptor, instead of controlling neuronal polarity as previously thought. Dishevelled (Dsh) proteins DSH-1 and MIG-5 redundantly mediate the repulsive activity of the Wnt signals to induce anterior outgrowth, whereas DSH-1 also provides feedback inhibition to attenuate the signaling to allow posterior outgrowth against the Wnt gradient. This inhibitory function of DSH-1, which requires its dishevelled, Egl-10, and pleckstrin (DEP) domain, acts by promoting LIN-17 phosphorylation and is antagonized by planar cell polarity signaling components Van Gogh (VANG-1) and Prickle (PRKL-1). Our results suggest that Dsh proteins both respond to Wnt signals to shape neuronal projections and moderate its activity to fine-tune neuronal morphology. PMID:26460008

  7. Coseeded Schwann cells myelinate neurites from differentiated neural stem cells in neurotrophin-3-loaded PLGA carriers.

    PubMed

    Xiong, Yi; Zhu, Ji-Xiang; Fang, Zheng-Yu; Zeng, Cheng-Guang; Zhang, Chao; Qi, Guo-Long; Li, Man-Hui; Zhang, Wei; Quan, Da-Ping; Wan, Jun

    2012-01-01

    Biomaterials and neurotrophic factors represent promising guidance for neural repair. In this study, we combined poly-(lactic acid-co-glycolic acid) (PLGA) conduits and neurotrophin-3 (NT-3) to generate NT-3-loaded PLGA carriers in vitro. Bioactive NT-3 was released stably and constantly from PLGA conduits for up to 4 weeks. Neural stem cells (NSCs) and Schwann cells (SCs) were coseeded into an NT-releasing scaffold system and cultured for 14 days. Immunoreactivity against Map2 showed that most of the grafted cells (>80%) were differentiated toward neurons. Double-immunostaining for synaptogenesis and myelination revealed the formation of synaptic structures and myelin sheaths in the coculture, which was also observed under electron microscope. Furthermore, under depolarizing conditions, these synapses were excitable and capable of releasing synaptic vesicles labeled with FM1-43 or FM4-64. Taken together, coseeding NSCs and SCs into NT-3-loaded PLGA carriers increased the differentiation of NSCs into neurons, developed synaptic connections, exhibited synaptic activities, and myelination of neurites by the accompanying SCs. These results provide an experimental basis that supports transplantation of functional neural construction in spinal cord injury. PMID:22619535

  8. Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans

    PubMed Central

    Zheng, Chaogu; Diaz-Cuadros, Margarete; Chalfie, Martin

    2015-01-01

    Wnt proteins regulate axonal outgrowth along the anterior–posterior axis, but the intracellular mechanisms that modulate the strength of Wnt signaling in axon guidance are largely unknown. Using the Caenorhabditis elegans mechanosensory PLM neurons, we found that posteriorly enriched LIN-44/Wnt acts as a repellent to promote anteriorly directed neurite outgrowth through the LIN-17/Frizzled receptor, instead of controlling neuronal polarity as previously thought. Dishevelled (Dsh) proteins DSH-1 and MIG-5 redundantly mediate the repulsive activity of the Wnt signals to induce anterior outgrowth, whereas DSH-1 also provides feedback inhibition to attenuate the signaling to allow posterior outgrowth against the Wnt gradient. This inhibitory function of DSH-1, which requires its dishevelled, Egl-10, and pleckstrin (DEP) domain, acts by promoting LIN-17 phosphorylation and is antagonized by planar cell polarity signaling components Van Gogh (VANG-1) and Prickle (PRKL-1). Our results suggest that Dsh proteins both respond to Wnt signals to shape neuronal projections and moderate its activity to fine-tune neuronal morphology. PMID:26460008

  9. Short Report: Olfactory Ensheathing Cells Promote Differentiation of Neural Stem Cells and Robust Neurite Extension

    PubMed Central

    Sethi, Rosh; Sethi, Roshan; Redmond, Andy

    2014-01-01

    Aims The goal of this study was to gain insight into the signaling between olfactory ensheathing cells (OECs) and neural stem cells (NSCs). We sought to understand the impact of OECs on NSC differentiation and neurite extension and to begin to elucidate the factors involved in these interactions to provide new targets for therapeutic interventions. Materials and Methods We utilized lines of OECs that have been extremely well characterized in vitro and in vivo along with well studied NSCs in gels to determine the impact of the coculture in three dimensions. To further elucidate the signaling, we used conditioned media from the OECs as well as fractioned components on NSCs to determine the molecular weight range of the soluble factors that was most responsible for the NSC behavior. Results We found that the coculture of NSCs and OECs led to robust NSC differentiation and extremely long neural processes not usually seen with NSCs in three dimensional gels in vitro. Through culture of NSCs with fractioned OEC media, we determined that molecules larger than 30 kDa have the greatest impact on the NSC behavior. Conclusions Overall, our findings suggest that cocultures of NSCs and OECs may be a novel combination therapy for neural injuries including spinal cord injury (SCI). Furthermore, we have identified a class of molecules which plays a substantial role in the behavior that provides new targets for investigating pharmacological therapies. PMID:24996386

  10. Cell patterning with a heptagon acoustic tweezer--application in neurite guidance.

    PubMed

    Gesellchen, F; Bernassau, A L; Déjardin, T; Cumming, D R S; Riehle, M O

    2014-07-01

    Accurate control over positioning of cells is a highly desirable feature in tissue engineering applications since it allows, for example, population of substrates in a controlled fashion, rather than relying on random seeding. Current methods to achieve a differential distribution of cells mostly use passive patterning methods to change chemical, mechanical or topographic properties of surfaces, making areas differentially permissive to the adhesion of cells. However, these methods have no ad hoc control over the actual deposition of cells. Direct patterning methods like bioprinting offer good control over cell position, but require sophisticated instrumentation and are often cost- and time-intensive. Here, we present a novel electronically controlled method of generating dynamic cell patterns by acoustic trapping of cells at a user-determined position, with a heptagonal acoustic tweezer device. We demonstrate the capability of the device to create complex patterns of cells using the device's ability to re-position acoustic traps by using a phase shift in the acoustic wave, and by switching the configuration of active piezoelectric transducers. Furthermore, we show that by arranging Schwann cells from neonatal rats in a linear pattern we are able to create Bands of Büngner-like structures on a non-structured surface and demonstrate that these features are able to guide neurite outgrowth from neonatal rat dorsal root ganglia. PMID:24817215

  11. Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones.

    PubMed

    Munnamalai, Vidhya; Weaver, Cory J; Weisheit, Corinne E; Venkatraman, Prahatha; Agim, Zeynep Sena; Quinn, Mark T; Suter, Daniel M

    2014-08-01

    NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones. PMID:24702317

  12. Neurite-specific Ca2+ dynamics underlying sound processing in an auditory interneurone.

    PubMed

    Baden, T; Hedwig, B

    2007-01-01

    Concepts on neuronal signal processing and integration at a cellular and subcellular level are driven by recording techniques and model systems available. The cricket CNS with the omega-1-neurone (ON1) provides a model system for auditory pattern recognition and directional processing. Exploiting ON1's planar structure we simultaneously imaged free intracellular Ca(2+) at both input and output neurites and recorded the membrane potential in vivo during acoustic stimulation. In response to a single sound pulse the rate of Ca(2+) rise followed the onset spike rate of ON1, while the final Ca(2+) level depended on the mean spike rate. Ca(2+) rapidly increased in both dendritic and axonal arborizations and only gradually in the axon and the cell body. Ca(2+) levels were particularly high at the spike-generating zone. Through the activation of a Ca(2+)-sensitive K(+) current this may exhibit a specific control over the cell's electrical response properties. In all cellular compartments presentation of species-specific calling song caused distinct oscillations of the Ca(2+) level in the chirp rhythm, but not the faster syllable rhythm. The Ca(2+)-mediated hyperpolarization of ON1 suppressed background spike activity between chirps, acting as a noise filter. During directional auditory processing, the functional interaction of Ca(2+)-mediated inhibition and contralateral synaptic inhibition was demonstrated. Upon stimulation with different sound frequencies, the dendrites, but not the axonal arborizations, demonstrated a tonotopic response profile. This mirrored the dominance of the species-specific carrier frequency and resulted in spatial filtering of high frequency auditory inputs. PMID:17443773

  13. Silver nanoparticles at sublethal concentrations disrupt cytoskeleton and neurite dynamics in cultured adult neural stem cells.

    PubMed

    Cooper, Robert J; Spitzer, Nadja

    2015-05-01

    Silver nanoparticles (AgNPs) have potent antimicrobial properties at concentrations far below those that cause cytotoxic and genotoxic effects in eukaryotic cells. This property has resulted in the widespread use of AgNPs in consumer products, leading to environmental exposures at sub-lethal levels through ingestion and inhalation. Although the toxicity of AgNPs has been well characterized, effects of environmentally relevant exposures have not been extensively investigated in spite of studies that suggest accumulation of silver in tissues, including brain. To assess the sublethal effects of AgNPs on neural cell function, we used cultured SVZ-NSCs, a model of neurogenesis and neural cells. Throughout life, neural stem cells (NSCs) in the subventricular zone (SVZ) of the lateral ventricles proliferate and migrate via the rostral migratory stream to the olfactory bulb. Once there, they complete differentiation into neurons and glia and integrate into existing circuits. This process of neurogenesis is tightly regulated, and is considered a part of healthy brain function. We found that 1.0 μg/mL AgNP exposure in cultured differentiating NSCs induced the formation of f-actin inclusions, indicating a disruption of actin function. These inclusions did not co-localize with AgNPs, and therefore do not represent sequestered nanoparticles. Further, AgNP exposure led to a reduction in neurite extension and branching in live cells, cytoskeleton-mediated processes vital to neurogenesis. We conclude that AgNPs at sublethal concentrations disrupt actin dynamics in SVZ-NSCs, and that an associated disruption in neurogenesis may contribute to documented deficits in brain function following AgNP exposure. PMID:25952507

  14. DBZ regulates cortical cell positioning and neurite development by sustaining the anterograde transport of Lis1 and DISC1 through control of Ndel1 dual-phosphorylation.

    PubMed

    Okamoto, Masayuki; Iguchi, Tokuichi; Hattori, Tsuyoshi; Matsuzaki, Shinsuke; Koyama, Yoshihisa; Taniguchi, Manabu; Komada, Munekazu; Xie, Min-Jue; Yagi, Hideshi; Shimizu, Shoko; Konishi, Yoshiyuki; Omi, Minoru; Yoshimi, Tomohiko; Tachibana, Taro; Fujieda, Shigeharu; Katayama, Taiichi; Ito, Akira; Hirotsune, Shinji; Tohyama, Masaya; Sato, Makoto

    2015-02-18

    Cell positioning and neuronal network formation are crucial for proper brain function. Disrupted-in-Schizophrenia 1 (DISC1) is anterogradely transported to the neurite tips, together with Lis1, and functions in neurite extension via suppression of GSK3β activity. Then, transported Lis1 is retrogradely transported and functions in cell migration. Here, we show that DISC1-binding zinc finger protein (DBZ), together with DISC1, regulates mouse cortical cell positioning and neurite development in vivo. DBZ hindered Ndel1 phosphorylation at threonine 219 and serine 251. DBZ depletion or expression of a double-phosphorylated mimetic form of Ndel1 impaired the transport of Lis1 and DISC1 to the neurite tips and hampered microtubule elongation. Moreover, application of DISC1 or a GSK3β inhibitor rescued the impairments caused by DBZ insufficiency or double-phosphorylated Ndel1 expression. We concluded that DBZ controls cell positioning and neurite development by interfering with Ndel1 from disproportionate phosphorylation, which is critical for appropriate anterograde transport of the DISC1-complex. PMID:25698733

  15. Minimum line width of ion beam-modified polystyrene by negative carbon ions for nerve-cell attachment and neurite extension

    NASA Astrophysics Data System (ADS)

    Sommani, P.; Tsuji, H.; Sato, H.; Kitamura, T.; Hattori, M.; Gotoh, Y.; Ishikawa, J.

    2007-04-01

    The minimum line width of the negative-ion-modified polystyrene (PS) for guidance and immobilizations of nerve-cell body and neurite extension have been investigated. Carbon negative ions were implanted into PS at fluence of 3 × 1015 ions/cm2 and energy of 5-20 keV through the various triangle apertures of the micro-pattern mask. After in vitro culture of the nerve-like cells of rat adrenal pheochromocytoma (PC12h), results showed that the minimum line widths for a single cell attachment and for neurite extension were 5-7 and 3-5 μm, respectively. While the minimum line width for attachment of cell group with long neurite was about 20 μm. The suitable widths for a large number of cells and for neurite extension were 20 and 5 μm, respectively. Therefore, the guidance for a clear separation of the attachment size of cell body and neurite extension could be achieved by the different modified line widths.

  16. CaMKII-Mediated CREB Phosphorylation Is Involved in Ca2+-Induced BDNF mRNA Transcription and Neurite Outgrowth Promoted by Electrical Stimulation.

    PubMed

    Yan, Xiaodong; Liu, Juanfang; Ye, Zhengxu; Huang, Jinghui; He, Fei; Xiao, Wei; Hu, Xueyu; Luo, Zhuojing

    2016-01-01

    Electrical stimulation (ES)-triggered up-regulation of brain-derived neurotrophic factor (BDNF) and neurite outgrowth in cultured rat postnatal dorsal root ganglion neurons (DRGNs) is calcium (Ca2+)-dependent. The effects of increased Ca2+ on BDNF up-regulation and neurite outgrowth remain unclear. We showed here that ES increased phosphorylation of the cAMP-response element binding protein (CREB). Blockade of Ca2+ suppressed CREB phosphorylation and neurite outgrowth. Down-regulation of phosphorylated (p)-CREB reduced BDNF transcription and neurite outgrowth triggered by ES. Furthermore, blockade of calmodulin-dependent protein kinase II (CaMKII) using the inhibitors KN93 or KN62 reduced p-CREB, and specific knockdown of the CaMKIIα or CaMKIIβ subunit was sufficient to suppress p-CREB. Recombinant BDNF or hyperforin reversed the effects of Ca2+ blockade and CaMKII knockdown. Taken together, these data establish a potential signaling pathway of Ca2+-CaMKII-CREB in neuronal activation. To our knowledge, this is the first report of the mechanisms of Ca2+-dependent BDNF transcription and neurite outgrowth triggered by ES. These findings might help further investigation of complex molecular signaling networks in ES-triggered nerve regeneration in vivo. PMID:27611779

  17. Early expression of glycine and GABA(A) receptors in developing spinal cord neurons. Effects on neurite outgrowth.

    PubMed

    Tapia, J C; Mentis, G Z; Navarrete, R; Nualart, F; Figueroa, E; Sánchez, A; Aguayo, L G

    2001-01-01

    Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABA(A)-activated currents were demonstrated during applications of glycine and GABA (50-100 microM) in 5 days in vitro (DIV) neurons. Long application (> or =10 min) of 100 microM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg(2+), at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca(2+) indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca(2+) events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABA(A) receptors provide a fundamental regulation of both

  18. Pure neuritic leprosy presenting as ulnar nerve neuropathy: a case report of electrodiagnostic, radiographic, and histopathological findings.

    PubMed

    Payne, Russell; Baccon, Jennifer; Dossett, John; Scollard, David; Byler, Debra; Patel, Akshal; Harbaugh, Kimberly

    2015-11-01

    Hansen's disease, or leprosy, is a chronic infectious disease with many manifestations. Though still a major health concern and leading cause of peripheral neuropathy in the developing world, it is rare in the United States, with only about 150 cases reported each year. Nevertheless, it is imperative that neurosurgeons consider it in the differential diagnosis of neuropathy. The causative organism is Mycobacterium leprae, which infects and damages Schwann cells in the peripheral nervous system, leading first to sensory and then to motor deficits. A rare presentation of Hansen's disease is pure neuritic leprosy. It is characterized by nerve involvement without the characteristic cutaneous stigmata. The authors of this report describe a case of pure neuritic leprosy presenting as ulnar nerve neuropathy with corresponding radiographic, electrodiagnostic, and histopathological data. This 11-year-old, otherwise healthy male presented with progressive right-hand weakness and numbness with no cutaneous abnormalities. Physical examination and electrodiagnostic testing revealed findings consistent with a severe ulnar neuropathy at the elbow. Magnetic resonance imaging revealed diffuse thickening and enhancement of the ulnar nerve and narrowing at the cubital tunnel. The patient underwent ulnar nerve decompression with biopsy. Pathology revealed acid-fast organisms within the nerve, which was pathognomonic for Hansen's disease. He was started on antibiotic therapy, and on follow-up he had improved strength and sensation in the ulnar nerve distribution. Pure neuritic leprosy, though rare in the United States, should be considered in the differential diagnosis of those presenting with peripheral neuropathy and a history of travel to leprosy-endemic areas. The long incubation period of M. leprae, the ability of leprosy to mimic other conditions, and the low sensitivity of serological tests make clinical, electrodiagnostic, and radiographic evaluation necessary for diagnosis

  19. Hormonal modulation of the quantity and in situ activity of tyrosine hydroxylase in neurites of the median eminence.

    PubMed Central

    Wang, P S; Porter, J C

    1986-01-01

    The role of ovarian hormones in the control of the quantity and activity of tyrosine hydroxylase (TyrOHase) in neurites of the median eminence of the rat was investigated. TyrOHase was quantified by an immunoblot assay using purified rat TyrOHase as the standard. Treatment of ovariectomized animals with progesterone, but not estradiol, resulted in a significant reduction in the amount of TyrOHase in the median eminence. The in situ activity of the enzyme was assayed by measuring the rate of synthesis of L-3,4-dihydroxyphenylalanine (dopa), and the results were expressed as mol of dopa per hr per mol of TyrOHase. In animals treated with both estradiol and progesterone for 3 days, the in situ activity of TyrOHase in the median eminence was 114 +/- 13.5 (mean +/- SEM) compared to 26 +/- 4.7 for the controls. Estradiol or progesterone alone was much less effective than was the combination of estradiol and progesterone. To ascertain whether the effect of estradiol and progesterone on TyrOHase activity was reflected in the secretion of dopamine into hypophyseal portal blood, ovariectomized rats were treated for 3 days with both estradiol and progesterone or with the solvent vehicle. The concentration of dopamine in portal plasma of the hormone-treated animals was 1.93 +/- 0.533 ng/ml compared to 0.34 +/- 0.094 ng/ml in vehicle-treated animals. We conclude that the quantity and in situ molar activity of TyrOHase in neurites of the median eminence as well as the secretion of dopamine from these neurites are modulated by the combined action of estradiol and progesterone. PMID:2879287

  20. Involvement of gecko SNAP25b in spinal cord regeneration by promoting outgrowth and elongation of neurites.

    PubMed

    Wang, Yingjie; Dong, Yingying; Song, Honghua; Liu, Yan; Liu, Mei; Yuan, Ying; Ding, Fei; Gu, Xiaosong; Wang, Yongjun

    2012-12-01

    SNARE complex mediates cellular membrane fusion events essential for neurotransmitter release and synaptogenesis. SNAP25, a member of the SNARE proteins, plays critical roles during the development of the central nervous system via regulation by alternative splicing and protein kinase phosphorylation. To date, little information is available regarding the protein in the spinal cord regeneration, especially for the postnatal highly expressed isoform SNAP25b. In the present study, we characterized gecko SNAP25b, which shared high identity with those of other vertebrates. Expression of gecko SNAP25b was temporally upregulated in both neurons of spinal cord and forming ependymal tube following tail amputation, coinciding with the occurrence of regenerate re-innervation. Overexpression of gecko wild type SNAP25b in the SH-SY5Y and undifferentiated PC12 cells promoted the elongation and outgrowth of neurites, while mutant constructs at Serine(187) resulted in differential effects for which S187A had a promoting role. Knockdown of endogenous SNAP25b affected the formation of neurites, which could be rescued by overexpression of SNAP25b. FM1-43 staining revealed that transfection of S187E mutant construct reduced the recruitment of vesicles. In addition, transfection of gecko SNAP25b in the astrocyte, which is absent from neuronal specific VAMP2, was capable of enhancing process elongation, indicating a potential for various alternative protein combinations. Taken together, our data suggest that gecko SNAP25b is involved in spinal cord regeneration by promoting outgrowth and elongation of neurites in a more extensive protein binding manner. PMID:23010346

  1. Role of Tropomodulin’s Leucine Rich Repeat Domain in the Formation of Neurite-like Processes

    PubMed Central

    2015-01-01

    Actin dynamics is fundamental for neurite development; monomer depolymerization from pointed ends is rate-limiting in actin treadmilling. Tropomodulins (Tmod) make up a family of actin pointed end-capping proteins. Of the four known isoforms, Tmod1–Tmod3 are expressed in brain cells. We investigated the role of Tmod’s C-terminal (LRR) domain in the formation of neurite-like processes by overexpressing Tmod1 and Tmod2 with deleted or mutated LRR domains in PC12 cells, a model system used to study neuritogenesis. Tmod1 overexpression results in a normal quantity and a normal length of processes, while Tmod2 overexpression reduces both measures. The Tmod2 overexpression phenotype is mimicked by overexpression of Tmod1 with the LRR domain removed or with three point mutations in the LRR domain that disrupt exposed clusters of conserved residues. Removal of Tmod2’s LRR domain does not significantly alter the outgrowth of neurite-like processes compared to that of Tmod2. Overexpression of chimeras with the N-terminal and C-terminal domains switched between Tmod1 and Tmod2 reinforces the idea that Tmod1’s LRR domain counteracts the reductive effect of the Tmod N-terminal domain upon formation of processes while Tmod2’s LRR domain does not. We suggest that the TM-dependent actin capping ability of both Tmods inhibits the formation of processes, but in Tmod1, this inhibition can be controlled via its LRR domain. Circular dichroism, limited proteolysis, and molecular dynamics demonstrate structural differences in the C-terminal region of the LRR domains of Tmod1, Tmod2, and the Tmod1 mutant. PMID:24746171

  2. Nanostructured Polyaniline Coating on ITO Glass Promotes the Neurite Outgrowth of PC 12 Cells by Electrical Stimulation.

    PubMed

    Wang, Liping; Huang, Qianwei; Wang, Jin-Ye

    2015-11-10

    A conducting polymer polyaniline (PANI) with nanostructure was synthesized on indium tin oxide (ITO) glass. The effect of electrical stimulation on the proliferation and the length of neurites of PC 12 cells was investigated. The dynamic protein adsorption on PANI and ITO surfaces in a cell culture medium was also compared with and without electrical stimulation. The adsorbed proteins were characterized using SDS-PAGE. A PANI coating on ITO surface was shown with 30-50 nm spherical nanostructure. The number of PC 12 cells was significantly greater on the PANI/ITO surface than on ITO and plate surfaces after cell seeding for 24 and 36 h. This result confirmed that the PANI coating is nontoxic to PC 12 cells. The electrical stimulation for 1, 2, and 4 h significantly enhanced the cell numbers for both PANI and ITO conducting surfaces. Moreover, the application of electrical stimulation also improved the neurite outgrowth of PC 12 cells, and the number of PC 12 cells with longer neurite lengths increased obviously under electrical stimulation for the PANI surface. From the mechanism, the adsorption of DMEM proteins was found to be enhanced by electrical stimulation for both PANI/ITO and ITO surfaces. A new band 2 (around 37 kDa) was observed from the collected adsorbed proteins when PC 12 cells were cultured on these surfaces, and culturing PC 12 cells also seemed to increase the amount of band 1 (around 90 kDa). When immersing PANI/ITO and ITO surfaces in a DMEM medium without a cell culture, the number of band 3 (around 70 kDa) and band 4 (around 45 kDa) proteins decreased compared to that of PC 12 cell cultured surfaces. These results are valuable for the design and improvement of the material performance for neural regeneration. PMID:25992643

  3. Effect of 710 nm visible light irradiation on neurite outgrowth in primary rat cortical neurons following ischemic insult

    SciTech Connect

    Choi, Dong-Hee; Lee, Kyoung-Hee; Kim, Ji-Hye; Kim, Moon Young; Lim, Jeong Hoon; Lee, Jongmin

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer 710 nm wavelength light (LED) has a protective effect in the stroke animal model. Black-Right-Pointing-Pointer We determined the effects of LED irradiation in vitro stroke model. Black-Right-Pointing-Pointer LED treatment promotes the neurite outgrowth through MAPK activation. Black-Right-Pointing-Pointer The level of synaptic markers significantly increased with LED treatment. Black-Right-Pointing-Pointer LED treatment protects cell death in the in vitro stroke model. -- Abstract: Objective: We previously reported that 710 nm Light-emitting Diode (LED) has a protective effect through cellular immunity activation in the stroke animal model. However, whether LED directly protects neurons suffering from neurodegeneration was entirely unknown. Therefore, we sought to determine the effects of 710 nm visible light irradiation on neuronal protection and neuronal outgrowth in an in vitro stroke model. Materials and methods: Primary cultured rat cortical neurons were exposed to oxygen-glucose deprivation (OGD) and reoxygenation and normal conditions. An LED array with a peak wavelength of 710 nm was placed beneath the covered culture dishes with the room light turned off and were irradiated accordingly. LED treatments (4 min at 4 J/cm{sup 2} and 50 mW/cm{sup 2}) were given once to four times within 8 h at 2 h intervals for 7 days. Mean neurite density, mean neurite diameter, and total fiber length were also measured after microtubule associated protein 2 (MAP2) immunostaining using the Axio Vision program. Synaptic marker expression and MAPK activation were confirmed by Western blotting. Results: Images captured after MAP2 immunocytochemistry showed significant (p < 0.05) enhancement of post-ischemic neurite outgrowth with LED treatment once and twice a day. MAPK activation was enhanced by LED treatment in both OGD-exposed and normal cells. The levels of synaptic markers such as PSD 95, GAP 43, and synaptophysin significantly

  4. Hericium erinaceus (Bull.: Fr) Pers. cultivated under tropical conditions: isolation of hericenones and demonstration of NGF-mediated neurite outgrowth in PC12 cells via MEK/ERK and PI3K-Akt signaling pathways.

    PubMed

    Phan, Chia-Wei; Lee, Guan-Serm; Hong, Sok-Lai; Wong, Yuin-Teng; Brkljača, Robert; Urban, Sylvia; Abd Malek, Sri Nurestri; Sabaratnam, Vikineswary

    2014-12-01

    Hericium erinaceus (Bull.: Fr.) Pers. is an edible and medicinal mushroom used traditionally to improve memory. In this study, we investigated the neuritogenic effects of hericenones isolated from H. erinaceus and the mechanisms of action involved. H. erinaceus was cultivated and the secondary metabolites were elucidated by high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR). The secondary metabolites were tested for neurite outgrowth activity (if any). Rat pheochromocytoma (PC12) cells were employed and the nerve growth factor (NGF) level was also determined. The signaling pathways involved in the mushroom-induced neuritogenesis were investigated using several pharmacological inhibitors. Hericenones B-E (1-4), erinacerin A (5) and isohericerin (6) were isolated from the basidiocarps of H. erinaceus. The hericenones did not promote neurite outgrowth but when induced with a low concentration of NGF (5 ng mL(-1)), the neuritogenic activity was comparable to that of the positive control (50 ng mL(-1) of NGF). Hericenone E was able to stimulate NGF secretion which was two-fold higher than that of the positive control. The neuritogenesis process was partially blocked by the tyrosine kinase receptor (Trk) inhibitor, K252a, suggesting that the neuritogenic effect was not solely due to NGF. Hericenone E also increased the phosphorylation of extracellular-signal regulated kinases (ER