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Sample records for early neurite outgrowth

  1. Munc18 and Munc13 regulate early neurite outgrowth

    PubMed Central

    Broeke, Jurjen H.P.; Roelandse, Martijn; Luteijn, Maartje J.; Boiko, Tatiana; Matus, Andrew; Toonen, Ruud F.; Verhage, Matthijs

    2010-01-01

    Background information. During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth. Results. We tested this possibility in dissociated cell cultures and organotypic slice cultures of two release-deficient mice (Munc18-1 null and Munc13-1/2 double null). Both types of release-deficient neurons have a decreased outgrowth speed and therefore have a smaller total neurite length during early development [DIV1–4 (day in vitro 1–4)]. In addition, more filopodia per growth cone were observed in Munc18-1 null, but not WT (wild-type) or Munc13-1/2 double null neurons. The smaller total neurite length during early development was no longer observed after synaptogenesis (DIV14–23). Conclusion. These data suggest that the inability of vesicle fusion in the growth cone affects outgrowth during the initial phases when outgrowth speed is high, but not during/after synaptogenesis. Overall, the outgrowth speed is probably not rate-limiting during neuronal network formation, at least in vitro. In addition, Munc18, but not Munc13, regulates growth cone filopodia, potentially via its previously observed effect on filamentous actin. PMID:20497124

  2. Tropomodulins are negative regulators of neurite outgrowth

    PubMed Central

    Fath, Thomas; Fischer, Robert S.; Dehmelt, Leif; Halpain, Shelley; Fowler, Velia M.

    2010-01-01

    Regulation of the actin cytoskeleton is critical for neurite formation. Tropomodulins (Tmods) regulate polymerization at actin filament pointed ends. Previous experiments using a mouse model deficient for the neuron specific isoform Tmod2 suggested a role for Tmods in neuronal function by impacting processes underlying learning and memory. However, the role of Tmods in neuronal function on the cellular level remains unknown. Immunofluorescence localization of the neuronal isoforms Tmod1 and Tmod2 in cultured rat primary hippocampal neurons revealed that Tmod1 is enriched along the proximal part of F-actin bundles in lamellipodia of spreading cells and in growth cones of extending neurites, while Tmod2 appears largely cytoplasmic. Functional analysis of these Tmod isoforms in a mouse neuroblastoma N2a cell line showed that knockdown of Tmod2 resulted in a significant increase in number of neurite-forming cells and in neurite length. While N2a cells compensated for Tmod2 knockdown by increasing Tmod1 levels, over-expression of exogenous Tmod1 had no effect on neurite outgrowth. Moreover, knockdown of Tmod1 increased the number of neurites formed per cell, without effect on number of neurite-forming cells or neurite length. Taken together, these results indicate that Tmod1 and Tmod2 have mechanistically distinct inhibitory roles in neurite formation, likely mediated via different effects on F-actin dynamics and via differential localizations during early neuritogenesis. PMID:21146252

  3. Characterization of BASP1-mediated neurite outgrowth.

    PubMed

    Korshunova, Irina; Caroni, Pico; Kolkova, Kateryna; Berezin, Vladimir; Bock, Elisabeth; Walmod, Peter S

    2008-08-01

    The brain acid-soluble protein BASP1 (CAP-23, NAP-22) belongs to the family of growth-associated proteins, which also includes GAP-43, a protein recently shown to regulate neural cell adhesion molecule (NCAM)-mediated neurite outgrowth. Here, the effects of BASP1 overexpression were investigated in PC12E2 cells and primary hippocampal neurons. BASP1 overexpression stimulated neurite outgrowth in both cell types. The effects of BASP1 and trans-homophilic NCAM interactions were additive, and BASP1-induced neurite outgrowth was not inhibited by ectopic expression of cytoplasmic NCAM domains. Furthermore, inhibition of signaling via the fibroblast growth factor receptor, Src-family nonreceptor tyrosine kinases, protein kinase C, or GSK3beta, and expression of constructs of the cytoskeletal proteins spectrin and tau inhibited NCAM- but not BASP1-induced neurite outgrowth. Expression of BASP1 mutated at the serine-5 phosphorylation site stimulated neurite outgrowth to a degree comparable to that observed in response to overexpression of wild-type BASP1, whereas expression of BASP1 mutated at the myristoylation site at glycine-1 completely abrogated the stimulatory effects of the protein on neurite outgrowth. Finally, coexpression experiments with dominant negative and wild-type versions of GAP-43 and BASP1 demonstrated that the two proteins could substitute for each other with respect to induction of NCAM-independent neurite outgrowth, whereas BASP1 was unable to replace the stimulatory effect of GAP-43 on NCAM-mediated neurite outgrowth. These observations demonstrate that BASP1 and GAP-43 have overlapping, but not identical, functions in relation to neurite outgrowth and indicate that the main function of BASP1 is to regulate the organization and morphology of the plasma membrane.

  4. Phosphate glass fibres promote neurite outgrowth and early regeneration in a peripheral nerve injury model.

    PubMed

    Kim, Young-Phil; Lee, Gil-Su; Kim, Jong-Wan; Kim, Min Soo; Ahn, Hong-Sun; Lim, Jae-Young; Kim, Hae-Won; Son, Young-Jin; Knowles, Jonathan C; Hyun, Jung Keun

    2015-03-01

    Three-dimensional (3D) scaffolds, which are bioactive and aid in neuronal guidance, are essential in the repair and regeneration of injured peripheral nerves. In this study, we used novel inorganic microfibres guided by phosphate glass (PG). PG fibres (PGfs) were aligned on compressed collagen that was rolled into a nerve conduit. In vitro tests confirmed that adult dorsal root ganglion (DRG) neurons showed active neurite outgrowth along the fibres, with a maximum number and length of neurites being significantly higher than those cultured on tissue culture plastic. In vivo experiments with nerve conduits that either contained PGfs (PGf/Col) or lacked them (Col) were conducted on transected sciatic nerves of rats for up to 12 weeks. One week after implantation, the PGf/Col group showed many axons extending along the scaffold, whereas the Col group showed none. Eight weeks after implantation, the PGf/Col group exhibited greater recovery of plantar muscle atrophy than the Col group. Electrophysiological studies revealed that some animals in the PGf/Col group at 6 and 7 weeks post-implantation (5.3% and 15.8%, respectively) showed compound muscle action potential. The Col group over the same period showed no response. Motor function also showed faster recovery in the PGf/Col group compared to the Col group up to 7 weeks. However, there was no significant difference in the number of axons, muscle atrophy or motor and sensory functions between the two groups at 12 weeks post-implantation. In summary, phosphate glass fibres can promote directional growth of axons in cases of peripheral nerve injury by acting as physical guides. Copyright © 2012 John Wiley & Sons, Ltd.

  5. Acetylcholinesterase modulates neurite outgrowth on fibronectin.

    PubMed

    Giordano, C; Poiana, G; Augusti-Tocco, G; Biagioni, S

    2007-05-04

    Acetylcholinesterase (AChE) has been reported to be involved in the modulation of neurite outgrowth. To understand the role played by different domains, we transfected neuroblastoma cells with three constructs containing the invariant region of AChE, differing in the exon encoding the C-terminus and therefore in AChE cellular fate and localization. All isoforms increased neurite extension, suggesting the involvement of the invariant domain [A. De Jaco, G. Augusti-Tocco, S. Biagioni, Alternative AChE molecular forms exhibit similar ability to induce neurite outgrowth, J. Neurosci. Res. 70 (2002) 756-765]. The peripheral anionic site (PAS) is encoded by invariant exons and represents the domain involved in non-cholinergic functions of AChE. Masking of PAS with fasciculin results in a significant decrease of neurite outgrowth in all clones overexpressing AChE. A strong reduction was also observed when clones were cultured on fibronectin. Treatment of clones with fasciculin, therefore masking PAS, abolished the fibronectin-induced reduction. The inhibition of the catalytic site cannot revert the fibronectin effect. Finally, when clones were cultured on fibronectin in the presence of heparin, a ligand of fibronectin, the inhibitory effect was completely reversed. Our results indicate that PAS could directly or indirectly mediate AChE/fibronectin interactions.

  6. Neurite outgrowth inhibitors in gliotic tissue.

    PubMed

    Nieto-Sampedro, M

    1999-01-01

    Gliotic tissue is the major obstacle to axon regeneration after CNS injury. We designed tissue culture assays to search for molecules responsible for neurite outgrowth inhibition in gliotic tissue. All the inhibitory activity in injured brain tissue was located in a plasma membrane heparan-sulphate and condroitin-sulphate type-proteoglycan of apparent molecular weight 200 kDalton. The proteoglycan core protein (apparent MW 48,000 kD) was biologically inactive, whereas the glycosamine-glycan (GAG) chains accounted for the inhibitory activity. Because of its cell location and mode of induction, the inhibitor was called injured membrane proteoglycan, IMP. IMP prevented neurite outgrowth initiation when attached to the culture substrate and caused growth cone collapse when added in solution to neurons with already growing neurites. We concluded that IMP was responsible for preventing injured CNS fibre regeneration. Double-staining immunohistochemistry of normal and gliotic tissue with anti-IMP monoclonal antibodies together with glial and neuronal markers, permitted the unequivocal definition of inhibitor presenting cells by confocal microscopy. IMP-immunostaining in normal CNS was observed exclusively on neurons. However, after a lesion, immunostaining occurred primarily on intensely GFAP-positive reactive astrocytes, but not on OX-42 positive microglia. The availability of antibodies permitted rapid affinity-purification of the neurite inhibitor and comparison with similar molecules possibly expressed during development. IMP itself or a highly related form, was expressed in embryonic brain, reaching maximal expression around postnatal day 3 and decreasing strongly in normal adult tissue. Perinatal rat brain proteoglycans inhibited neurite outgrowth similarly, though not identically, to IMP. Our data suggest that perinatal membrane and injured membrane proteoglycans may differ in GAG composition. IMP-like immunoreactivity was also found in developing brain

  7. 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.

  8. 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.

  9. 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.

  10. CRMP-5 interacts with actin to regulate neurite outgrowth

    PubMed Central

    GONG, XIAOBING; TAN, MINGHUI; GAO, YUAN; CHEN, KEEN; GUO, GUOQING

    2016-01-01

    CRMP family proteins (CRMPs) are abundantly expressed in the developing nervous system mediating growth cone guidance, neuronal polarity and axon elongation. CRMP-5 has been indicated to serve a critical role in neurite outgrowth. However, the detailed mechanisms of how CRMP-5 regulates neurite outgrowth remain unclear. In the current study, co-immunoprecipitation was used to identify the fact that CRMP-5 interacted with the actin and tubulin cytoskeleton networks in the growth cones of developing hippocampal neurons. CRMP-5 exhibited increased affinity towards actin when compared with microtubules. Immunocytochemistry was used to identify the fact that CRMP-5 colocalized with actin predominantly in the C-domain and T-zone in growth cones. In addition, genetic inhibition of CRMP-5 by siRNA suppressed the expression of actin, growth cone development and neurite outgrowth. Overexpression of CRMP-5 promoted the interaction with actin, growth cone development and hippocampal neurite outgrowth. Taken together, these data suggest that CRMP-5 is able to interact with the actin cytoskeleton network in the growth cone and affect growth cone development and neurite outgrowth via this interaction in developing hippocampal neurons. PMID:26677106

  11. Rab22 controls NGF signaling and neurite outgrowth in PC12 cells.

    PubMed

    Wang, Liang; Liang, Zhimin; Li, Guangpu

    2011-10-01

    Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22-Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22-Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.

  12. 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.

  13. Automatic quantification of neurite outgrowth by means of image analysis

    NASA Astrophysics Data System (ADS)

    Van de Wouwer, Gert; Nuydens, Rony; Meert, Theo; Weyn, Barbara

    2004-07-01

    A system for quantification of neurite outgrowth in in-vitro experiments is described. The system is developed for routine use in a high-throughput setting and is therefore needs fast, cheap, and robust. It relies on automated digital microscopical imaging of microtiter plates. Image analysis is applied to extract features for characterisation of neurite outgrowth. The system is tested in a dose-response experiment on PC12 cells + Taxol. The performance of the system and its ability to measure changes on neuronal morphology is studied.

  14. 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

  15. 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

  16. 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

  17. Shoc2/Sur8 protein regulates neurite outgrowth.

    PubMed

    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.

  18. The transcription factor ATF-3 promotes neurite outgrowth.

    PubMed

    Seijffers, Rhona; Allchorne, Andrew J; Woolf, Clifford J

    2006-01-01

    Dorsal root ganglion (DRG) neurons regenerate after a peripheral nerve injury but not after injury to their axons in the spinal cord. A key question is which transcription factors drive the changes in gene expression that increase the intrinsic growth state of peripherally injured sensory neurons? A prime candidate is activating transcription factor-3 (ATF-3), a transcription factor that we find is induced in all DRG neurons after peripheral, but not central axonal injury. Moreover, we show in adult DRG neurons that a preconditioning peripheral, but not central axonal injury, increases their growth, correlating closely with the pattern of ATF-3 induction. Using viral vectors, we delivered ATF-3 to cultured adult DRG neurons and find that ATF-3 enhances neurite outgrowth. Furthermore, ATF-3 promotes long sparsely branched neurites. ATF-3 overexpression did not increase c-Jun expression. ATF-3 may contribute, therefore, to neurite outgrowth by orchestrating the gene expression responses in injured neurons.

  19. Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons

    PubMed Central

    Park, Han-A; Licznerski, Pawel; Alavian, Kambiz N.; Shanabrough, Marya

    2015-01-01

    Abstract Aims: B-cell lymphoma-extra large (Bcl-xL) protects survival in dividing cells and developing neurons, but was not known to regulate growth. Growth and synapse formation are indispensable for neuronal survival in development, inextricably linking these processes. We have previously shown that, during synaptic plasticity, Bcl-xL produces changes in synapse number, size, activity, and mitochondrial metabolism. In this study, we determine whether Bcl-xL is required for healthy neurite outgrowth and whether neurite outgrowth is necessary for survival in developing neurons in the presence or absence of stress. Results: Depletion of endogenous Bcl-xL impairs neurite outgrowth in hippocampal neurons followed by delayed cell death which is dependent on upregulation of death receptor 6 (DR6), a molecule that regulates axonal pruning. Under hypoxic conditions, Bcl-xL-depleted neurons demonstrate increased vulnerability to neuronal process loss and to death compared with hypoxic controls. Endogenous DR6 expression and upregulation during hypoxia are associated with worsened neurite damage; depletion of DR6 partially rescues neuronal process loss, placing DR6 downstream of the effects of Bcl-xL on neuronal process outgrowth and protection. In vivo ischemia produces early increases in DR6, suggesting a role for DR6 in brain injury. Innovation: We suggest that DR6 levels are usually suppressed by Bcl-xL; Bcl-xL depletion leads to upregulation of DR6, failure of neuronal outgrowth in nonstressed cells, and exacerbation of hypoxia-induced neuronal injury. Conclusion: Bcl-xL regulates neuronal outgrowth during development and protects neurites from hypoxic insult, as opposed by DR6. Factors that enhance neurite formation may protect neurons against hypoxic injury or neurodegenerative stimuli. Antioxid. Redox Signal. 22, 93–108. PMID:24787232

  20. Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension

    PubMed Central

    Shepard, Jaclyn A.; Stevans, Alyson C.; Holland, Samantha; Wang, Christine E.; Shikanov, Ariella; Shea, Lonnie D.

    2012-01-01

    Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration. PMID:22038654

  1. Neurite outgrowth in human iPSC-derived neurons

    EPA Pesticide Factsheets

    Data on morphology of rat and human neurons in cell cultureThis dataset is associated with the following publication:Druwe, I., T. Freudenrich , K. Wallace , T. Shafer , and W. Mundy. Comparison of Human Induced PluripotentStem Cell-Derived Neurons and Rat Primary CorticalNeurons as In Vitro Models of Neurite Outgrowth. Applied In vitro Toxicology. Mary Ann Liebert, Inc., Larchmont, NY, USA, 2(1): 26-36, (2016).

  2. 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.

  3. Neurite Outgrowth at the Biomimetic Interface

    PubMed Central

    Kofron, Celinda M.; Liu, Yu-Ting; López-Fagundo, Cristina Y.; Mitchel, Jennifer A.; Hoffman-Kim, Diane

    2010-01-01

    Understanding the cues that guide axons and how we can optimize these cues to achieve directed neuronal growth is imperative for neural tissue engineering. Cells in the local environment influence neurons with a rich combination of cues. This study deconstructs the complex mixture of guidance cues by working at the biomimetic interface - isolating the topographical information presented by cells and determining its capacity to guide neurons. We generated replica materials presenting topographies of oriented astrocytes (ACs), endothelial cells (ECs), and Schwann cells (SCs) as well as computer-aided design materials inspired by the contours of these cells (bioinspired-CAD). These materials presented distinct topographies and anisotropies and in all cases were sufficient to guide neurons. Dorsal root ganglia (DRG) cells and neurites demonstrated the most directed response on bioinspired-CAD materials which presented anisotropic features with 90° edges. DRG alignment was strongest on SC bioinspired-CAD materials followed by AC bioinspired-CAD materials, with more uniform orientation to EC bioinspired-CAD materials. Alignment was strongest on SC replica materials followed by AC and EC replicas. These results suggest that the topographies of anisotropic tissue structures are sufficient for neuronal guidance. This work is discussed in the context of feature dimensions, morphology, and guidepost hypotheses. PMID:20440561

  4. Na+/Ca2+ exchanger inhibitors inhibit neurite outgrowth in PC12 cells.

    PubMed

    Oda, Toru; Kume, Toshiaki; Izumi, Yasuhiko; Ishihara, Kumatoshi; Sugmimoto, Hachiro; Akaike, Akinori

    2011-01-01

    To elucidate the role of Na(+)/Ca(2+) exchanger (NCX) in neurite outgrowth, we investigated the effects of NCX inhibitors on neurite outgrowth in PC12 cells. KB-R7943 and 3',4'-dichlorobenzamil, NCX inhibitors, inhibited the neurite outgrowth caused by nerve growth factor (NGF). NCX inhibitors inhibited the neurite outgrowth caused by dibutylyl cAMP, which rapidly reorganizes the cytoskeleton. KB-R7943 inhibited the neurite outgrowth caused by Y-27632, an inhibitor of Rho kinase (ROCK) that regulates actin. However, NCX inhibitors did not inhibit NGF-induced phosphorylation of extracellular signal-regulated kinase. These results suggest that NCX inhibitor affects downstream of the Rho-ROCK signal transduction pathways in neurite outgrowth.

  5. Sonic Hedgehog Promotes Neurite Outgrowth of Primary Cortical Neurons Through Up-Regulating BDNF Expression.

    PubMed

    He, Weiliang; Cui, Lili; Zhang, Cong; Zhang, Xiangjian; He, Junna; Xie, Yanzhao

    2016-04-01

    Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary cortical neurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary cortical neurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary cortical neurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary cortical neurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary cortical neurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary cortical neurons at least partially through modulating BDNF expression.

  6. Astroglial differentiation is required for support of neurite outgrowth.

    PubMed

    Wang, L C; Baird, D H; Hatten, M E; Mason, C A

    1994-05-01

    Models of astrocyte differentiation stress a lineage program that involves a progressive loss of astroglial support of neuronal differentiation. These models predict that astroglial promotion of neurite extension declines with the "age" of the astrocyte. An alternative view is that astroglial support of neurite growth is regulated by epigenetic factors that induce the cells either to differentiate and support neuronal functions or to undergo cell proliferation and fail to support neurons. To compare the contribution of astroglial cell "age" to astroglial support of neurite extension, mouse cerebellar astroglia were maintained in vitro for 3-90 d, and assayed for their ability to support neurite formation. When cultured in isolation, astroglial support of neurite extension declined with time in vitro, as assayed by quantifying outgrowth from explants of pontine nuclei, falling from a robust level just after the astroglia were harvested to negligible levels 21-90 d later. Since previous studies have shown that neurons can change the state of astroglial cells (Hatten, 1985), we tested the neurite promoting activity of astroglia that were cultured for 21-90 d in vitro and subsequently induced to differentiate by the addition of neurons. When granule neurons were added to aged astroglia and pontine explants plated 2 d later, neurite growth from the explants was exuberant, regardless of the time astroglia spent in vitro prior to the addition of neurons. The state of astroglia that were growth promoting or growth inhibiting was examined by bromodeoxyuridine staining and with antisera to glial filament protein. Aged astroglia cultured alone and thus inhibitory to axon growth, proliferated at high rates and had polygonal shapes. In contrast, aged astroglia to which neurons had been added, proliferated at low rates and developed process-bearing stellate shapes. To test further whether proliferation levels related to the growth-supporting properties of astroglia, astroglia

  7. Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells

    PubMed Central

    1985-01-01

    The outgrowth of neurites from rat PC12 cells stimulated by combined treatment of nerve growth factor (NGF) with cAMP is significantly more rapid and extensive than the outgrowth induced by either factor alone. We have compared the responses of PC12 cells under three different growth conditions, NGF alone, cAMP alone, and combined treatment, with respect to surface morphology, rapidity of neurite outgrowth, and stability of neurite microtubules, to understand the synergistic action of NGF and cAMP on PC12. Surface events at early times in these growth conditions varied, suggesting divergent pathways of action of NGF and cAMP. This suggestion is strongly supported by the finding that cells exposed to saturating levels of dibutyryl cAMP without substantial neurite outgrowth initiated neurites within 5 min of NGF. This response has been adopted as a convenient assay for NGF. Neurites that regenerated in the three growth conditions showed marked differences in stability to treatments that depolymerize microtubules. The results indicate that microtubules in cells treated with both NGF and cAMP are significantly more stable than in either growth factor alone. We suggest that a shift of the assembly equilibrium favoring tubulin assembly is a necessary prerequisite for the initiation of neurites by PC12. PMID:2982887

  8. 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.

  9. Rho kinase regulates neurite outgrowth of hippocampal neurons via calcium dependent cytoskeleton regulation

    PubMed Central

    Ji, Zhisheng; Cai, Zhenbin; Zhang, Jifeng; Liu, Nannuan; Chen, Jing; Tan, Minghui; Lin, Hongsheng; Guo, Guoqing

    2017-01-01

    Objective: To investigate whether calcium is involved in downstream signal transduction in neurite outgrowth regulated by Rho kinase. Methods: In vitro primary hippocampal neurons were cultured and treated with Rho kinase agonist (LPA) or antagonist (Y-27632). Then, the cytoskeleton and neurite outgrowth were observed. After addition of calcium antagonist BAPTA/AM to reduce intracellular calcium, the cytoskeleton distribution and neurite outgrowth were observed. Results: The activation or inhibition of Rho kinase could significantly alter the number and length of neurites of hippocampal neurons. Rho kinase regulated the cytoskeleton to regulate the neurite outgrowth, and LPA could significantly increase intracellular calcium. After BAPTA/AM treatment, the length and branch number of neurites of neurons reduced markedly. BAPTA/AM was able to reduce intracellular calcium and decrease neuronal cytoskeleton. Treatment with both BAPTA/AM and LPA could stop the retraction of neurites, but the length and branch number of neurites remained unchanged after treatment with Y-27632 and LPA. Conclusion: Calcium may affect the cytoskeleton arrangement to regulate neurite outgrowth, and calcium is involved in the downstream signal transduction of Rho kinase regulated neurite outgrowth of hippocampal neurons. PMID:28337305

  10. Neurite outgrowth on cultured spiral ganglion neurons induced by erythropoietin.

    PubMed

    Berkingali, Nurdanat; Warnecke, Athanasia; Gomes, Priya; Paasche, Gerrit; Tack, Jan; Lenarz, Thomas; Stöver, Timo

    2008-09-01

    The morphological correlate of deafness is the loss of hair cells with subsequent degeneration of spiral ganglion neurons (SGN). Neurotrophic factors have a neuroprotective effect, and especially brain-derived neurotrophic factor (BDNF) has been demonstrated to protect SGN in vitro and after ototoxic trauma in vivo. Erythropoietin (EPO) attenuates hair cell loss in rat cochlea explants that were treated with gentamycin. Recently, it has also been shown that EPO reduces the apoptose rate in hippocampal neurons. Therefore, the aim of the study was to examine the effects of EPO on SGN in vitro. Spiral ganglion cells were isolated from neonatal rats and cultured for 48 h in serum-free medium supplemented with EPO and/or BDNF. Results showed that survival rates of SGN were not significantly improved when cultivated with EPO alone. Also, EPO did not further increase BDNF-induced survival of SGN. However, significant elongation of neurites was determined when SGN were cultivated with EPO alone. Even though a less than additive effect was observed, combined treatment with BDNF and EPO led to a significant elongation of neurites when compared to individual treatment with BDNF or EPO. It can be concluded that EPO induces neurite outgrowth rather than promoting survival. Thus, EPO presents as an interesting candidate to enhance and modulate the regenerative effect of BDNF on SGN.

  11. Oriented Schwann cell monolayers for directed neurite outgrowth.

    PubMed

    Thompson, Deanna M; Buettner, Helen M

    2004-08-01

    Schwann cells are an important component of the peripheral nervous system and participate in peripheral nerve regeneration. They create a supportive environment for neurite outgrowth by releasing trophic factors and up-regulating permissive molecules on their surface. In addition, Schwann cells are able to self-organize into linear arrays in vitro and in vivo, suggesting a possible role in neurite guidance. Previously, we showed that Schwann cell placement and orientation in subconfluent cultures can be controlled using microlithographically patterned laminin substrates (Thompson, D. M., and H. M. Buettner. Tissue Eng. 7(3):247-266, 2001). In the current study, these substrates were used to create oriented Schwann cell monolayers. Both Schwann cell orientation and coverage were quantified in response to seeding density, culture medium, and micropattern dimensions. In serum-free medium, increasing the seeding density yielded a linear increase in coverage of the substrate area but decreased cell alignment. In an alternate approach, Schwann cells were first seeded in serum-free medium at moderate seeding density, allowed to align, then expanded in serum-containing growth medium. This produced complete coverage without large seeding densities while preserving alignment to the micropattern. Alignment and coverage were unaffected by micropattern dimensions. This work provides a useful methodology for investigating Schwann cell guidance effects on growing neurites.

  12. Staurosporin induces neurite outgrowth through ROS generation in HN33 hippocampal cell lines.

    PubMed

    Min, J Y; Park, M H; Park, M K; Park, K W; Lee, N W; Kim, T; Kim, H J; Lee, D H

    2006-11-01

    Staurosporin, a specific inhibitor of PKC, is widely used in studies of signal transduction pathways. Previous studies have shown that staurosporin induces neurite outgrowth, but the underlying mechanisms remain unclear. Here we report that staurosporin induces neurite outgrowth in HN33 hippocampal cells. Two other PKC inhibitors, Go 6976 (specific for alpha- and beta-isoforms) and rotterlin (a selective inhibitor of PKC delta), have no neuritogenic effect. In addition, staurosporin specifically increases ROS generation. NAC, which inhibits the generation of ROS, suppresses the staurosporin-induced neurite outgrowth in HN33 cells. Further, H(2)O(2) causes neurite outgrowth. Taken together, these results confirm a neuritogenic effect of staurosporin and point to ROS as the signal mediator of staurosporin-induced neurite outgrowth in HN33 hippocampal cells. Theme: Development and regeneration Topic: Neurotrophic factors: receptors and cellular mechanisms.

  13. Neurite outgrowth is driven by actin polymerization even in the presence of actin polymerization inhibitors

    PubMed Central

    Chia, Jonathan X.; Efimova, Nadia; Svitkina, Tatyana M.

    2016-01-01

    Actin polymerization is a universal mechanism to drive plasma membrane protrusion in motile cells. One apparent exception to this rule is continuing or even accelerated outgrowth of neuronal processes in the presence of actin polymerization inhibitors. This fact, together with the key role of microtubule dynamics in neurite outgrowth, led to the concept that microtubules directly drive plasma membrane protrusion either in the course of polymerization or by motor-driven sliding. The possibility that unextinguished actin polymerization drives neurite outgrowth in the presence of actin drugs was not explored. We show that cultured hippocampal neurons treated with cytochalasin D or latrunculin B contained dense accumulations of branched actin filaments at ∼50% of neurite tips at all tested drug concentrations (1–10 μM). Actin polymerization is required for neurite outgrowth because only low concentrations of either inhibitor increased the length and/or number of neurites, whereas high concentrations inhibited neurite outgrowth. Of importance, neurites undergoing active elongation invariably contained a bright F-actin patch at the tip, whereas actin-depleted neurites never elongated, even though they still contained dynamic microtubules. Stabilization of microtubules by Taxol treatment did not stop elongation of cytochalasin–treated neurites. We conclude that actin polymerization is indispensable for neurite elongation. PMID:27682586

  14. Micropatterning of Neurite Outgrowth in vitro Using Micropipette Drawing

    NASA Astrophysics Data System (ADS)

    Goto, Miho; Moriguchi, Hiroyuki; Takayama, Yuzo; Kotani, Kiyoshi; Jimbo, Yasuhiko

    To understand the relationship between neuronal-network functions and single-neuron activity, construction of artificial neuronal network is one of the promising approaches. Cell patterning is a useful technique to get single-neuron-based networks in vitro. Here in this work, we propose a simple method to get simple neuronal networks, based on neurite-outgrowth guidance. Our method, referred to as “micropipette drawing” is a quite simple photomask-free technique. Growth-guiding patterns are drawn with a micropipette containing cell-adhesive solution on non-adhesive substrates. Guiding structures of approximately 10 μm width were successfully drawn and rat hippocampal neurons were cultured on the patterns. The patterned neuronal networks could be maintained for more than a week.

  15. Repeated, intermittent treatment with amphetamine induces neurite outgrowth in rat pheochromocytoma cells (PC12 cells).

    PubMed

    Park, Yang Hae; Kantor, Lana; Wang, Kevin K W; Gnegy, Margaret E

    2002-09-27

    Repeated, intermittent treatment with amphetamine (AMPH) leads to long-term neurobiological adaptations in rat brain including an increased number and branching of dendritic spines. This effect depends upon several different cell types in the intact brain. Here we demonstrate that repeated, intermittent AMPH treatment induces neurite outgrowth in cultured PC12 cells without the requirement for integrated synaptic pathways. PC12 cells were treated with 1 micro M AMPH for 5 min a day, for 5 days. After 10 days of withdrawal, there was an increase in the percentage of cells with neurites ( approximately 30%) and the length of neurites as well as an increase in the level of GAP-43 and neurofilament-M. Neurite outgrowth was enhanced as withdrawal time was increased. Neurite outgrowth was much greater following repeated, intermittent treatment with AMPH compared to continuous or single treatment with AMPH. Pretreatment with cocaine, a monoamine transporter blocker, inhibited the AMPH-mediated increase in neurite outgrowth. Neither NGF antibody nor DA receptor antagonists blocked AMPH-induced neurite outgrowth, demonstrating that AMPH-induced neurite outgrowth is not dependent on endogenous NGF release or DA receptors. Thus we have demonstrated that repeated, intermittent treatment with AMPH has a neurotrophic effect in PC12 cells. The effect requires the action of AMPH on the norepinephrine transporter, and shares characteristics in its development with other forms of sensitization but does not require an intact neuroanatomy.

  16. 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.

  17. 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

  18. 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.

  19. Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

    PubMed

    Kumar, Sanjay; Chakraborty, Saikat; Barbosa, Cindy; Brustovetsky, Tatiana; Brustovetsky, Nickolay; Obukhov, Alexander G

    2012-04-01

    Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPCs changes significantly during brain development, suggesting that fine-tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)-differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up-regulated TRPC1 and TRPC6 expression, but down-regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF-induced neurite outgrowth. Conversely, shRNA-mediated knockdown of TRPC1 decreased, whereas shRNA-mediated knockdown of TRPC5 increased NGF-induced neurite extension. Endogenous TRPC1 attenuated the anti-neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1-TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN-TRPC6) facilitated neurite outgrowth in NGF-differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF-differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up-regulated TRPC1 and TRPC6 expression via a p75(NTR)-IKK(2)-dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up-regulated TRPC1 and TRPC6 via an IKK(2) dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF-dependent "molecular damper" maintaining a submaximal velocity of neurite extension.

  20. Contact-associated neurite outgrowth and branching of immature cortical interneurons.

    PubMed

    Sang, Qian; Tan, Seong-Seng

    2003-06-01

    When juvenile interneurons arrive at the cortical environment following tangential migration, they are faced with the task of positioning themselves in cortical space in preparation for local circuit wiring. This includes integration into different cortical layers and cessation of migration at various positions to ensure adequate coverage. Little is known about the signals or mechanisms that initiate a conversion from the migratory phenotype to the arborization phenotype. This study looks at the immediate changes in interneuron morphology after culturing for 24 h in a three-dimensional collagen gel. Immature interneurons taken from different stages of corticogenesis showed increased neurite branching and outgrowth after interneuronal contacts were made. These responses were suppressed in the presence of Slit and brain-derived neurotrophic factor (BDNF) if the interneurons were sourced from early to mid-stages of corticogenesis. However, interneurons taken from the late period of corticogenesis responded to Slit and BDNF by increasing branching and neurite outgrowth. These results suggest an initial interneuronal cell contact as a stimulus for propagating neuronal arborization that may lead to the formation of inhibitory neuronal circuits. In addition, we have identified the late corticogenetic period when interneurons are most sensitive to the neurite promoting effects of Slit and BDNF.

  1. 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...

  2. 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...

  3. Effect of viscosity on neurite outgrowth and fractal dimension.

    PubMed

    Caserta, F; Hausman, R E; Eldred, W D; Kimmel, C; Stanley, H E

    1992-03-02

    The growth mechanism by which neurons achieve their characteristic ramified morphology has long been of interest, but determining whether physical parameters, such as viscosity, are important has been difficult due to a lack of useful hypotheses and standard reproducible techniques. We have recently shown that neurons exhibit fractal behavior and that their fractal dimension (df) is consistent with a physical process called diffusion-limited aggregation (DLA). We suggested that this DLA behavior might stem from viscosity differences, chemical gradients or electrical fields (Caserta et al., Phys. Rev. Lett., 64 (1990) 95-98). DLA is a model for a large family of growth processes. In order for a process to fit the DLA model, the growth rate must be proportional to the gradient of a field at a point on the growing structure (Feder, Plenum, New York, 1988, Ch. 4). Chemical, electrical, or fluid pressure fields can fit the model depending on the particular physical system under study. Here, we studied growth of retinal neurons from chick embryos in culture media of various fluid viscosities. Thus, we test whether DLA in this system was based on a fluid pressure field. As viscosity was increased from 1 to 4.3 cps, the number of neurite branches decreased 98%. However, there was no effect on df. Over this range of viscosities, total cellular protein synthesis decreased only 17%. The results indicate that, while differences in viscosity between the interior and exterior of the cell affect neurite outgrowth, they do not affect the fractal behavior of neurons. Thus, viscosity differences are not the basis for the DLA pattern of neuronal arborization.

  4. 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

  5. The influence of magnetic fields exposure on neurite outgrowth in PC12 rat pheochromocytoma cells

    NASA Astrophysics Data System (ADS)

    Fan, W.; Ding, J.; Duan, W.; Zhu, Y. M.

    2004-11-01

    The aim of present work was to investigate the influence of magnetic fields exposure on neurite outgrowth in PC12 cells. The neurite number per cell, length of neurites and directions of neurite growth with respect to the direction of the magnetic field were analyzed after exposure to 50 Hz electromagnetic field for 96 h. A promotion was observed under a weak field (0.23 mT), as the average number of neurites per cell increased to 2.38±0.06 compared to 1.91±0.07 neurites/cell of the control dishes, while inhibition and directional outgrowth was evident under a relatively stronger field (1.32 mT). Our work shows that biological systems can be very sensitive to the strength of electromagnetic field.

  6. Serotonin (5-HT) regulates neurite outgrowth through 5-HT1A and 5-HT7 receptors in cultured hippocampal neurons.

    PubMed

    Rojas, Paulina S; Neira, David; Muñoz, Mauricio; Lavandero, Sergio; Fiedler, Jenny L

    2014-08-01

    Serotonin (5-HT) production and expression of 5-HT receptors (5-HTRs) occur early during prenatal development. Recent evidence suggests that, in addition to its classical role as a neurotransmitter, 5-HT regulates neuronal connectivity during mammalian development by modulating cell migration and neuronal cytoarchitecture. Given the variety of 5-HTRs, researchers have had difficulty clarifying the specific role of each receptor subtype in brain development. Signalling mediated by the G-protein-coupled 5-HT1A R and 5-HT7 R, however, has been associated with neuronal plasticity. Thus, we hypothesized that 5-HT promotes neurite outgrowth through 5-HT1A R and 5-HT7 R. The involvement of 5-HT1A R and 5-HT7 R in the morphology of rat hippocampal neurons was evaluated by treating primary cultures at 2 days in vitro with 5-HT and specific antagonists for 5-HT1A R and 5-HT7 R (WAY-100635 and SB269970, respectively). The stimulation of hippocampal neurons with 100 nM 5-HT for 24 hr produced no effect on either the number or the length of primary neurites. Nonetheless, after 5HT7 R was blocked, the addition of 5-HT increased the number of primary neurites, suggesting that 5HT7 R could inhibit neuritogenesis. In contrast, 5-HT induced secondary neurite outgrowth, an effect inhibited by 1 μM WAY-100635 or SB269970. These results suggest that both serotonergic receptors participate in secondary neurite outgrowth. We conclude that 5-HT1A R and 5-HT7 R regulate neuronal morphology in primary hippocampal cultures by promoting secondary neurite outgrowth.

  7. SRRM4-dependent neuron-specific alternative splicing of protrudin transcripts regulates neurite outgrowth

    PubMed Central

    Ohnishi, Takafumi; Shirane, Michiko; Nakayama, Keiichi I.

    2017-01-01

    Alternative splicing gives rise to diversity of the proteome, and it is especially prevalent in the mammalian nervous system. Indeed, many factors that control the splicing process govern nervous system development. Among such factors, SRRM4 is an important regulator of aspects of neural differentiation including neurite outgrowth. The mechanism by which SRRM4 regulates neurite outgrowth has remained poorly understood, however. We now show that SRRM4 regulates the splicing of protrudin gene (Zfyve27) transcripts in neuronal cells. SRRM4 was found to promote splicing of protrudin pre-mRNA so as to include a microexon (exon L) encoding seven amino acids in a neuron-specific manner. The resulting protein (protrudin-L) promotes neurite outgrowth during neurogenesis. Depletion of SRRM4 in Neuro2A cells impaired inclusion of exon L in protrudin mRNA, resulting in the generation of a shorter protein isoform (protrudin-S) that is less effective at promoting neurite extension. SRRM4 was found to recognize a UGC motif that is located immediately upstream of exon L and is necessary for inclusion of exon L in the mature transcript. Deletion of exon L in Neuro2A or embryonic stem cells inhibited neurite outgrowth. Our results suggest that SRRM4 controls neurite outgrowth through regulation of alternative splicing of protrudin transcripts. PMID:28106138

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

    PubMed

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

    2016-02-23

    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.

  9. Design of Hyaluronic Acid Hydrogels to Promote Neurite Outgrowth in Three Dimensions.

    PubMed

    Tarus, Dominte; Hamard, Lauriane; Caraguel, Flavien; Wion, Didier; Szarpak-Jankowska, Anna; van der Sanden, Boudewijn; Auzély-Velty, Rachel

    2016-09-28

    A hyaluronic acid (HA)-based extracellular matrix (ECM) platform with independently tunable stiffness and density of cell-adhesive peptide (RGD, arginine-glycine-aspartic acid) that mimics key biochemical and mechanical features of brain matrix has been designed. We demonstrated here its utility in elucidating ECM regulation of neural progenitor cell behavior and neurite outgrowth. The analysis of neurite outgrowth in 3-D by two-photon microscopy showed several important results in the development of these hydrogels. First, the ability of neurites to extend deeply into these soft HA-based matrices even in the absence of cell-adhesive ligand further confirms the potential of HA hydrogels for central nervous system (CNS) regeneration. Second, the behavior of hippocampal neural progenitor cells differed markedly between the hydrogels with a storage modulus of 400 Pa and those with a modulus of 800 Pa. We observed an increased outgrowth and density of neurites in the softest hydrogels (G' = 400 Pa). Interestingly, cells seeded on the surface of the hydrogels functionalized with the RGD ligand experienced an optimum in neurite outgrowth as a function of ligand density. Surprinsingly, neurites preferentially progressed inside the gels in a vertical direction, suggesting that outgrowth is directed by the hydrogel structure. This work may provide design principles for the development of hydrogels to facilitate neuronal regeneration in the adult brain.

  10. 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

  11. Tiam1 as a signaling mediator of nerve growth factor-dependent neurite outgrowth.

    PubMed

    Shirazi Fard, Shahrzad; Kele, Julianna; Vilar, Marçal; Paratcha, Gustavo; Ledda, Fernanda

    2010-03-19

    Nerve Growth Factor (NGF)-induced neuronal differentiation requires the activation of members of the Rho family of small GTPases. However, the molecular mechanisms through which NGF regulates cytoskeletal changes and neurite outgrowth are not totally understood. In this work, we identify the Rac1-specific guanine exchange factor (GEF) Tiam1 as a novel mediator of NGF/TrkA-dependent neurite elongation. In particular, we report that knockdown of Tiam1 causes a significant reduction in Rac1 activity and neurite outgrowth induced by NGF. Physical interaction between Tiam1 and active Ras (Ras-GTP), but not tyrosine phosphorylation of Tiam1, plays a central role in Rac1 activation by NGF. In addition, our findings indicate that Ras is required to associate Tiam1 with Rac1 and promote Rac1 activation upon NGF stimulation. Taken together, these findings define a novel molecular mechanism through which Tiam1 mediates TrkA signaling and neurite outgrowth induced by NGF.

  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. Diazinon and diazoxon impair the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons

    PubMed Central

    Pizzurro, Daniella M.; Dao, Khoi; Costa, Lucio G.

    2014-01-01

    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, a 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. PMID:24342266

  14. Retinoic acid induces neurite outgrowth and growth cone turning in invertebrate neurons.

    PubMed

    Dmetrichuk, Jennifer M; Carlone, Robert L; Spencer, Gaynor E

    2006-06-01

    Identification of molecules involved in neurite outgrowth during development and/or regeneration is a major goal in the field of neuroscience. Retinoic acid (RA) is a biologically important metabolite of vitamin A that acts as a trophic factor and has been implicated in neurite outgrowth and regeneration in many vertebrate species. Although abundant in the CNS of many vertebrates, the precise role of RA in neural regeneration has yet to be determined. Moreover, very little information is available regarding the role of RA in invertebrate nervous systems. Here, we demonstrate for the first time that RA induces neurite outgrowth from invertebrate neurons. Using individually identified neurons isolated from the CNS of Lymnaea stagnalis, we demonstrated that a significantly greater proportion of cells produced neurite outgrowth in RA. RA also extended the duration of time that cells remained electrically excitable in vitro, and we showed that exogenously applied RA acted as a chemoattractive factor and induced growth cone turning toward the source of RA. This is the first demonstration that RA can induce turning of an individual growth cone. These data strongly suggest that the actions of RA on neurite outgrowth and cell survival are highly conserved across species.

  15. A Facile Method for Simultaneously Measuring Neuronal Cell Viability and Neurite Outgrowth

    PubMed Central

    K. Hancock, Michael; Kopp, Leisha; Kaur, Navjot; Hanson, Bonnie J.

    2015-01-01

    Neurite outgrowth is an important morphological phenotype of neuronal cells that correlates with their function and cell health, yet there are limited methods available for measuring this phenomenon. Current approaches to measuring neurite outgrowth are laborious and time-consuming, relying largely upon immunocytochemical staining of neuronal markers (e.g., beta-III tubulin or MAP2) followed by manual or automated microscopy for image acquisition and analysis. Here we report the development of a quick and simple dual-color fluorescent dye-based staining method that allows for the simultaneous measurement of neuronal cell health and relative neurite outgrowth from the same sample. An orangered fluorescent dye that stains cell membrane surfaces is used as an indirect reporter of changes in relative neurite outgrowth due to alterations in the number or length of membrane projections emanating from neuronal cell bodies. Cell viability is assessed simultaneously via the use of a cell-permeant dye that is converted by intracellular esterase activity from a non-fluorescent substrate to a green-fluorescent product. Using Neuroscreen-1 cells (a PC-12 subclone), primary rat cortex neurons, and human induced pluripotent stem cell (iPSC)-derived neurons, we demonstrate that this multiplex assay allows for rapid visualization and unbiased, quantitative plate reader analysis of neuronal cell health and neurite outgrowth. PMID:25853055

  16. 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.

  17. Triggering of high-speed neurite outgrowth using an optical microheater.

    PubMed

    Oyama, Kotaro; Zeeb, Vadim; Kawamura, Yuki; Arai, Tomomi; Gotoh, Mizuho; Itoh, Hideki; Itabashi, Takeshi; Suzuki, Madoka; Ishiwata, Shin'ichi

    2015-11-16

    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.

  18. CHLORHEXIDINE INHIBITS L1 CELL ADHESION MOLECULE MEDIATED NEURITE OUTGROWTH IN VITRO

    PubMed Central

    Milstone, Aaron M.; Bamford, Penny; Aucott, Susan W.; Tang, Ningfeng; White, Kimberly R.; Bearer, Cynthia F.

    2013-01-01

    Background Chlorhexidine is a skin disinfectant that reduces skin and mucous membrane bacterial colonization and inhibits organism growth. Despite numerous studies assessing chlorhexidine safety in term infants, residual concerns have limited its use in hospitalized neonates, especially low birth weight preterm infants. The aim of this study was to assess the potential neurotoxicity of chlorhexidine on the developing central nervous system using a well-established in vitro model of neurite outgrowth that includes laminin and L1 cell adhesion molecule (L1) as neurite outgrowth promoting substrates. Methods Cerebellar granule neurons are plated on either poly L-lysine, L1 or laminin. Chlorhexidine, hexachlorophene or their excipients are added to the media. Neurons are grown for 24 h, then fixed and neurite length measured. Results Chlorhexidine significantly reduced the length of neurites grown on L1 but not laminin. Chlorhexidine concentrations as low as 125 ng/ml statistically significantly reduced neurite length on L1. Hexachlorophene did not affect neurite length. Conclusion Chlorhexidine at concentrations detected in the blood following topical applications in preterm infants specifically inhibited L1 mediated neurite outgrowth of cerebellar granule neurons. It is now vital to determine whether the blood brain barrier is permeable to chlorhexidine in preterm infants. PMID:24126818

  19. Mouse Acetylcholinesterase Enhances Neurite Outgrowth of Rat R28 Cells Through Interaction With Laminin-1

    PubMed Central

    Sperling, Laura E.; Klaczinski, Janine; Schütz, Corina; Rudolph, Lydia; Layer, Paul G.

    2012-01-01

    The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert ‘non-classical’, morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1. PMID:22570738

  20. Long non-coding RNA Malat1 promotes neurite outgrowth through activation of ERK/MAPK signalling pathway in N2a cells.

    PubMed

    Chen, Lei; Feng, Peimin; Zhu, Xi; He, Shixu; Duan, Jialan; Zhou, Dong

    2016-11-01

    Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are playing critical roles in neurogenesis, yet the underlying molecular mechanisms remain largely elusive. Neurite outgrowth is an early step in neuronal differentiation and regeneration. Using in vitro differentiation of neuroblastoma-derived Neuro-2a (N2a) cell as a model, we performed expression profiling to identify lncRNAs putatively relevant for neurite outgrowth. We identified that Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was one of the most significantly up-regulated lncRNAs during N2a cell differentiation. Malat1 knockdown resulted in defects in neurite outgrowth as well as enhanced cell death. To pinpoint signalling pathways perturbed by Malat1 depletion, we then performed a reporter-based screening to examine the activities of 50 signalling pathways in Malat1 knockdown cells. We found that Malat1 knockdown resulted in conspicuous inhibition of Mitogen-Activated Protein Kinase (MAPK) signaling pathway as well as abnormal activation of Peroxisome proliferator-activated receptor (PPAR) and P53 signalling pathway. Inhibition of ERK/MAPK pathway with PD98059 potently blocked N2a cell neurite outgrowth, whereas phorbol 12-myristate 13-acetate-induced ERK activation rescued defects in neurite outgrowth and cell death induced by Malat1 depletion. Together, our results established a critical role of Malat1 in the early step of neuronal differentiation through activating ERK/MAPK signalling pathway.

  1. Arylsulfatase B modulates neurite outgrowth via astrocyte chondroitin-4-sulfate: dysregulation by ethanol.

    PubMed

    Zhang, Xiaolu; Bhattacharyya, Sumit; Kusumo, Handojo; Goodlett, Charles R; Tobacman, Joanne K; Guizzetti, Marina

    2014-02-01

    In utero ethanol exposure causes fetal alcohol spectrum disorders, associated with reduced brain plasticity; the mechanisms of these effects are not well understood, particularly with respect to glial involvement. Astrocytes release factors that modulate neurite outgrowth. We explored the hypothesis that ethanol inhibits neurite outgrowth by increasing the levels of inhibitory chondroitin sulfate proteoglycans (CSPGs) in astrocytes. Astrocyte treatment with ethanol inhibited the activity of arylsulfatase B (ARSB), the enzyme that removes sulfate groups from chondroitin-4-sulfate (C4S) and triggers the degradation of C4S, increased total sulfated glycosaminoglycans (GAGs), C4S, and neurocan core-protein content and inhibited neurite outgrowth in neurons cocultured with ethanol-treated astrocytes in vitro, effects reversed by treatment with recombinant ARSB. Ethanol also inhibited ARSB activity and increased sulfate GAG and neurocan levels in the developing hippocampus after in vivo ethanol exposure. ARSB silencing increased the levels of sulfated GAGs, C4S, and neurocan in astrocytes and inhibited neurite outgrowth in cocultured neurons, indicating that ARSB activity directly regulates C4S and affects neurocan expression. In summary, this study reports two major findings: ARSB modulates sulfated GAG and neurocan levels in astrocytes and astrocyte-mediated neurite outgrowth in cocultured neurons; and ethanol inhibits the activity of ARSB, increases sulfated GAG, C4S, and neurocan levels, and thereby inhibits astrocyte-mediated neurite outgrowth. An unscheduled increase in CSPGs in the developing brain may lead to altered brain connectivity and to premature decrease in neuronal plasticity and therefore represents a novel mechanism by which ethanol can exert its neurodevelopmental effects.

  2. Progesterone Antagonism of Neurite Outgrowth Depends on Microglial Activation via Pgrmc1/S2R

    PubMed Central

    Bali, N; Arimoto, J. M.; Morgan, T. E.

    2013-01-01

    Neuronal plasticity is regulated by the ovarian steroids estradiol (E2) and progesterone (P4) in many normal brain functions, as well as in acute response to injury and chronic neurodegenerative disease. In a female rat model of axotomy, the E2-dependent compensatory neuronal sprouting is antagonized by P4. To resolve complex glial-neuronal cell interactions, we used the “wounding-in-a-dish” model of neurons cocultured with astrocytes or mixed glia (microglia to astrocytes, 1:3). Although both astrocytes and mixed glia supported E2-enhanced neurite outgrowth, P4 antagonized E2-induced neurite outgrowth only with mixed glia, but not astrocytes alone. We now show that P4-E2 antagonism of neurite outgrowth is mediated by microglial expression of progesterone receptor (Pgr) membrane component 1 (Pgrmc1)/S2R, a putative nonclassical Pgr mediator with multiple functions. The P4-E2 antagonism of neurite outgrowth was restored by add-back of microglia to astrocyte-neuron cocultures. Because microglia do not express the classical Pgr, we examined the role of Pgrmc1, which is expressed in microglia in vitro and in vivo. Knockdown by siRNA-Pgrmc1 in microglia before add-back to astrocyte-neuron cocultures suppressed the P4-E2 antagonism of neurite outgrowth. Conditioned media from microglia restored the P4-E2 activity, but only if microglia were activated by lipopolysaccharide or by wounding. Moreover, the microglial activation was blocked by Pgmrc1-siRNA knockdown. These findings explain why nonwounded cultures without microglial activation lack P4 antagonism of E2-induced neurite outgrowth. We suggest that microglial activation may influence brain responses to exogenous P4, which is a prospective therapy in traumatic brain injury. PMID:23653459

  3. 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

  4. DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

    PubMed

    Won, Jong Hoon; Ahn, Kyong Hoon; Back, Moon Jung; Ha, Hae Chan; Jang, Ji Min; Kim, Ha Hyung; Choi, Sang-Zin; Son, Miwon; Kim, Dae Kyong

    2015-01-01

    In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

  5. Rosiglitazone promotes neurite outgrowth and mitochondrial function in N2A cells via PPARgamma pathway.

    PubMed

    Chiang, Ming-Chang; Cheng, Yi-Chuan; Chen, Han-Min; Liang, Yao-Jen; Yen, Chia-Hui

    2014-01-01

    Several pieces of evidence indicate that peroxisome proliferator-activated receptor gamma (PPARγ) stimulation promotes neuronal differentiation. However, to date, the effects of a synthetic PPARγ agonist (Rosiglitazone, Rosi) on neurite outgrowth have not yet been well described. Here we have evaluated the effects of Rosi on neurite outgrowth and mitochondrial function in the mouse neuroblastoma Neuro 2a (N2A) cell line. Our results show that Rosi promotes neurite outgrowth of N2A cells and significantly increases the population of neurite-bearing cells, with apparent increase of intracellular calcium and the expression of calmodulin-dependent kinase I (CaMKI). Rosi also increases the intracellular cAMP and expression of both protein kinase A (PKA) and cAMP response element binding protein (CREB). Phosphorylation of CREB was also detected in the Rosi treated N2A cells. Moreover, Rosi significantly increases the transcription of AMP-activated kinase (AMPK) and Sirtuin 1 (SIRT1). Besides, the expression of PPAR coactivator 1α (PGC1α), as well as the mRNA level its downstream genes, including nuclear respiratory factors 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (Tfam) were induced by Rosi treatments. Furthermore, Rosi increases the level of ATP, D-loop, and mitochondrial mass in N2A cells. Collectively, these findings provide an array of evidence that PPARγ activation provides beneficial neuronal networks within neurite outgrowth.

  6. 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.

  7. Neurite outgrowth in cultured mouse pelvic ganglia - Effects of neurotrophins and bladder tissue.

    PubMed

    Ekman, Mari; Zhu, Baoyi; Swärd, Karl; Uvelius, Bengt

    2017-07-01

    Neurotrophic factors regulate survival and growth of neurons. The urinary bladder is innervated via both sympathetic and parasympathetic neurons located in the major pelvic ganglion. The aim of the present study was to characterize the effects of the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) on the sprouting rate of sympathetic and parasympathetic neurites from the female mouse ganglion. The pelvic ganglion was dissected out and attached to a petri dish and cultured in vitro. All three factors (BDNF, NT-3 and NGF) stimulated neurite outgrowth of both sympathetic and parasympathetic neurites although BDNF and NT-3 had a higher stimulatory effect on parasympathetic ganglion cells. The neurotrophin receptors TrkA, TrkB and TrkC were all expressed in neurons of the ganglia. Co-culture of ganglia with urinary bladder tissue, but not diaphragm tissue, increased the sprouting rate of neurites. Active forms of BDNF and NT-3 were detected in urinary bladder tissue using western blotting whereas tissue from the diaphragm expressed NGF. Neurite outgrowth from the pelvic ganglion was inhibited by a TrkB receptor antagonist. We therefore suggest that the urinary bladder releases trophic factors, including BDNF and NT-3, which regulate neurite outgrowth via activation of neuronal Trk-receptors. These findings could influence future strategies for developing pharmaceuticals to improve re-innervation due to bladder pathologies. Copyright © 2017. Published by Elsevier B.V.

  8. Mesenchymal stem cell-derived exosomes from different sources selectively promote neuritic outgrowth.

    PubMed

    Lopez-Verrilli, M A; Caviedes, A; Cabrera, A; Sandoval, S; Wyneken, U; Khoury, M

    2016-04-21

    Mesenchymal stem cells (MSCs) obtained from bone marrow (BM) have been shown to promote neuronal growth and survival. However, the comparative effects of MSCs of different sources, including menstrual MSCs (MenSCs), BM, umbilical cord and chorion stem cells on neurite outgrowth have not yet been explored. Moreover, the modulatory effects of MSCs may be mediated by paracrine mechanisms, i.e. by molecules contained in the MSC secretome that includes soluble factors and extracellular vesicles such as microvesicles and/or exosomes. The biogenesis of microvesicles, characterized by a vesicle diameter of 50 to 1000 nm, involves membrane shedding while exosomes, of 30 to 100 nm in diameter, originate in the multivesicular bodies within cells. Both vesicle types, which can be harvested from the conditioned media of cell cultures by differential centrifugation steps, regulate the function of target cells due to their molecular content of microRNA, mRNA, proteins and lipids. Here, we compared the effect of human menstrual MSCs (MenSCs) mediated by cell-cell contact, by their total secretome or by secretome-derived extracellular vesicles on neuritic outgrowth in primary neuronal cultures. The contact of MenSCs with cortical neurons inhibited neurite outgrowth while their total secretome enhanced it. The extracellular vesicle fractions showed a distinctive effect: while the exosome-enriched fraction enhanced neurite outgrowth, the microvesicle-enriched fraction displayed an inhibitory effect. When we compared exosome fractions of different human MSC sources, MenSC exosomes showed superior effects on the growth of the longest neurite in cortical neurons and had a comparable effect to BM-SC exosomes on neurite outgrowth in dorsal root ganglia neurons. Thus, the growth-stimulating effects of exosomes derived from MenSCs as well as the opposing effects of both extracellular vesicle fractions provide important information regarding the potential use of MenSCs as therapeutic

  9. 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.

  10. 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...

  11. APP independent and dependent effects on neurite outgrowth are modulated by the receptor associated protein, RAP

    PubMed Central

    Billnitzer, Andrew J.; Barskaya, Irina; Yin, Cailing; Perez, Ruth G.

    2013-01-01

    Amyloid precursor protein (APP) and its secreted form, sAPP, contribute to the development of neurons in hippocampus, a brain region critical for learning and memory. Full-length APP binds the low-density lipoprotein receptor-related protein (LRP), which stimulates APP endocytosis. LRP also contributes to neurite growth. Furthermore, the receptor associated protein (RAP) binds LRP in a manner that blocks APP-LRP interactions. To elucidate APP contributions to neurite growth for full-length APP and sAPP, we cultured wild type (WT) and APP knockout (KO) neurons in sAPPα and/or RAP and measured neurite outgrowth at 1 day in vitro. Our data reveal that WT neurons had less axonal outgrowth including less axon branching. RAP treatment potentiated the inhibitory effects of APP. KO neurons had significantly more outgrowth and branching, especially in response to RAP, effects which were also associated with ERK2 activation. Our results affirm a major inhibitory role by full-length APP on all aspects of axonal and dendritic outgrowth, and show that RAP-LRP binding stimulated axon growth independently of APP. These findings support a major role for APP as an inhibitor of neurite growth and reveal novel signaling functions for LRP that may be disrupted by Alzheimer’s pathology or therapies aimed at APP processing. PMID:23061396

  12. APP independent and dependent effects on neurite outgrowth are modulated by the receptor associated protein (RAP).

    PubMed

    Billnitzer, Andrew J; Barskaya, Irina; Yin, Cailing; Perez, Ruth G

    2013-01-01

    Amyloid precursor protein (APP) and its secreted form, sAPP, contribute to the development of neurons in hippocampus, a brain region critical for learning and memory. Full-length APP binds the low-density lipoprotein receptor-related protein (LRP), which stimulates APP endocytosis. LRP also contributes to neurite growth. Furthermore, the receptor associated protein (RAP) binds LRP in a manner that blocks APP-LRP interactions. To elucidate APP contributions to neurite growth for full-length APP and sAPP, we cultured wild type (WT) and APP knockout (KO) neurons in sAPPα and/or RAP and measured neurite outgrowth at 1 day in vitro. Our data reveal that WT neurons had less axonal outgrowth including less axon branching. RAP treatment potentiated the inhibitory effects of APP. KO neurons had significantly more outgrowth and branching, especially in response to RAP, effects which were also associated with ERK2 activation. Our results affirm a major inhibitory role by full-length APP on all aspects of axonal and dendritic outgrowth, and show that RAP-LRP binding stimulated axon growth independently of APP. These findings support a major role for APP as an inhibitor of neurite growth and reveal novel signaling functions for LRP that may be disrupted by Alzheimer's pathology or therapies aimed at APP processing. © 2012 International Society for Neurochemistry.

  13. 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...

  14. The role of bioactive compounds on the promotion of neurite outgrowth.

    PubMed

    More, Sandeep Vasant; Koppula, Sushruta; Kim, In-Su; Kumar, Hemant; Kim, Byung-Wook; Choi, Dong-Kug

    2012-06-04

    Neurite loss is one of the cardinal features of neuronal injury. Apart from neuroprotection, reorganization of the lost neuronal network in the injured brain is necessary for the restoration of normal physiological functions. Neuritogenic activity of endogenous molecules in the brain such as nerve growth factor is well documented and supported by scientific studies which show innumerable compounds having neurite outgrowth activity from natural sources. Since the damaged brain lacks the reconstructive capacity, more efforts in research are focused on the identification of compounds that promote the reformation of neuronal networks. An abundancy of natural resources along with the corresponding activity profiles have shown promising results in the field of neuroscience. Recently, importance has also been placed on understanding neurite formation by natural products in relation to neuronal injury. Arrays of natural herbal products having plentiful active constituents have been found to enhance neurite outgrowth. They act synergistically with neurotrophic factors to promote neuritogenesis in the diseased brain. Therefore use of natural products for neuroregeneration provides new insights in drug development for treating neuronal injury. In this study, various compounds from natural sources with potential neurite outgrowth activity are reviewed in experimental models.

  15. Expression of a chimeric CSF1R-LTK mediates ligand-dependent neurite outgrowth.

    PubMed

    Yamada, Shigeru; Nomura, Takashi; Takano, Kota; Fujita, Satoshi; Miyake, Masato; Miyake, Jun

    2008-11-19

    In an earlier screening, we identified several genes for kinases that might control the extension of neurites. One of these genes encoded a leukocyte tyrosine kinase (LTK), which is a receptor tyrosine kinase whose ligands remain to be identified. To examine the possible role of this LTK in neurite outgrowth, we constructed a chimeric receptor, in which the extracellular domain of the receptor for colony-stimulating factor-1 was fused to the cytoplasmic domain of LTK, which allowed the selective activation of LTK by colony-stimulating factor-1. Our studies using this chimeric receptor suggest that activation of the tyrosine kinase activity of LTK is sufficient to promote neurite outgrowth through pathways that include reactions catalyzed by phosphatidylinositol 3-kinase and MAPK.

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

    PubMed

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

    1994-04-12

    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.

  17. Phospholipase Cdelta3 regulates RhoA/Rho kinase signaling and neurite outgrowth.

    PubMed

    Kouchi, Zen; Igarashi, Takahiro; Shibayama, Nami; Inanobe, Shunichi; Sakurai, Kazuyuki; Yamaguchi, Hideki; Fukuda, Toshifumi; Yanagi, Shigeru; Nakamura, Yoshikazu; Fukami, Kiyoko

    2011-03-11

    Phospholipase Cδ3 (PLCδ3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLCδ3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLCδ3 in neuronal migration and outgrowth. PLCδ3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLCδ3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in the cerebral cortex. In addition, PLCδ3KD inhibits axon and dendrite outgrowth in primary cortical neurons. PLCδ3KD also suppresses neurite formation of Neuro2a neuroblastoma cells induced by serum withdrawal or treatment with retinoic acid. This inhibition is released by the reintroduction of wild-type PLCδ3. Interestingly, the H393A mutant lacking phosphatidylinositol 4,5-bisphosphate hydrolyzing activity generates supernumerary protrusions, and a constitutively active mutant promotes extensive neurite outgrowth, indicating that PLC activity is important for normal neurite outgrowth. The introduction of dominant negative RhoA (RhoA-DN) or treatment with Y-27632, a Rho kinase-specific inhibitor, rescues the neurite extension in PLCδ3KD Neuro2a cells. Similar effects were also detected in primary cortical neurons. Furthermore, the RhoA expression level was significantly decreased by serum withdrawal or retinoic acid in control cells, although this decrease was not observed in PLCδ3KD cells. We also found that exogenous expression of PLCδ3 down-regulated RhoA protein, and constitutively active PLCδ3 promotes the RhoA down-regulation more significantly than PLCδ3 upon differentiation. These results indicate that PLCδ3 negatively regulates RhoA expression, inhibits RhoA/Rho kinase signaling, and thereby promotes neurite extension.

  18. 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

  19. Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization.

    PubMed Central

    Shea, T B; Beermann, M L

    1994-01-01

    The respective roles of neurofilaments (NFs), microtubules (MTs), and the microtubule-associated proteins (MAPs) MAP 1B and tau on neurite outgrowth and stabilization were probed by the intracellular delivery of specific antisera into transiently permeabilized NB2a/d1 cells during treatment with dbcAMP. Intracellular delivery of antisera specific for the low (NF-L), middle (NF-M), or extensively phosphorylated high (NF-H) molecular weight subunits did not prevent initial neurite elaboration, nor did it induce retraction of existing neurites elaborated by cells that had been previously treated for 1 d with dbcAMP. By contrast, intracellular delivery of antisera directed against tubulin reduced the percentage of cells with neurites at both these time points. Intracellular delivery of anti-NF-L and anti-NF-M antisera did not induce retraction in cells treated with dbcAMP for 3 d. However, intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, tau, or tubulin induced similar levels of neurite retraction at this time. Intracellular delivery of monoclonal antibodies (RT97 or SMI-31) directed against phosphorylated NF-H induced neurite retraction in cell treated with dbcAMP for 3 d; a monoclonal antibody (SMI-32) directed against nonphosphorylated NF-H did not induce neurite retraction at this time. By contrast, none of the above antisera induced retraction of neurites in cells treated with dbcAMP for 7 d. Neurites develop resistance to retraction by colchicine, first detectable in some neurites after 3 d and in the majority of neurites after 7 d of dbcAMP treatment. We therefore examined whether or not colchicine resistance was compromised by intracellular delivery of the above antisera. Colchicine treatment resulted in rapid neurite retraction after intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, or tau into cells that had previously been treated with dbcAMP for 7 d. By contrast, colchicine

  20. 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.

  1. Tiam1 as a Signaling Mediator of Nerve Growth Factor-Dependent Neurite Outgrowth

    PubMed Central

    Vilar, Marçal; Paratcha, Gustavo; Ledda, Fernanda

    2010-01-01

    Nerve Growth Factor (NGF)-induced neuronal differentiation requires the activation of members of the Rho family of small GTPases. However, the molecular mechanisms through which NGF regulates cytoskeletal changes and neurite outgrowth are not totally understood. In this work, we identify the Rac1-specific guanine exchange factor (GEF) Tiam1 as a novel mediator of NGF/TrkA-dependent neurite elongation. In particular, we report that knockdown of Tiam1 causes a significant reduction in Rac1 activity and neurite outgrowth induced by NGF. Physical interaction between Tiam1 and active Ras (Ras-GTP), but not tyrosine phosphorylation of Tiam1, plays a central role in Rac1 activation by NGF. In addition, our findings indicate that Ras is required to associate Tiam1 with Rac1 and promote Rac1 activation upon NGF stimulation. Taken together, these findings define a novel molecular mechanism through which Tiam1 mediates TrkA signaling and neurite outgrowth induced by NGF. PMID:20333299

  2. Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase.

    PubMed

    Tornieri, Karine; Welshhans, Kristy; Geddis, Matthew S; Rehder, Vincent

    2006-04-01

    Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.

  3. Cyclic AMP stimulates neurite outgrowth of lamprey reticulospinal neurons without substantially altering their biophysical properties.

    PubMed

    Pale, T; Frisch, E B; McClellan, A D

    2013-08-15

    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. 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

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

    PubMed

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

    2014-08-01

    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. 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. 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. 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.

  6. 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.

  7. 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

  8. Stimulation of neurite outgrowth using an electrically conducting polymer

    PubMed Central

    Schmidt, Christine E.; Shastri, Venkatram R.; Vacanti, Joseph P.; Langer, Robert

    1997-01-01

    Damage to peripheral nerves often cannot be repaired by the juxtaposition of the severed nerve ends. Surgeons have typically used autologous nerve grafts, which have several drawbacks including the need for multiple surgical procedures and loss of function at the donor site. As an alternative, the use of nerve guidance channels to bridge the gap between severed nerve ends is being explored. In this paper, the electrically conductive polymer—oxidized polypyrrole (PP)—has been evaluated for use as a substrate to enhance nerve cell interactions in culture as a first step toward potentially using such polymers to stimulate in vivo nerve regeneration. Image analysis demonstrates that PC-12 cells and primary chicken sciatic nerve explants attached and extended neurites equally well on both PP films and tissue culture polystyrene in the absence of electrical stimulation. In contrast, PC-12 cells interacted poorly with indium tin oxide (ITO), poly(l-lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) surfaces. However, PC-12 cells cultured on PP films and subjected to an electrical stimulus through the film showed a significant increase in neurite lengths compared with ones that were not subjected to electrical stimulation through the film and tissue culture polystyrene controls. The median neurite length for PC-12 cells grown on PP and subjected to an electrical stimulus was 18.14 μm (n = 5643) compared with 9.5 μm (n = 4440) for controls. Furthermore, animal implantation studies reveal that PP invokes little adverse tissue response compared with poly(lactic acid-co-glycolic acid). PMID:9256415

  9. Stimulation of Neurite Outgrowth Using an Electrically Conducting Polymer

    NASA Astrophysics Data System (ADS)

    Schmidt, Christine E.; Shastri, Venkatram R.; Vacanti, Joseph P.; Langer, Robert

    1997-08-01

    Damage to peripheral nerves often cannot be repaired by the juxtaposition of the severed nerve ends. Surgeons have typically used autologous nerve grafts, which have several drawbacks including the need for multiple surgical procedures and loss of function at the donor site. As an alternative, the use of nerve guidance channels to bridge the gap between severed nerve ends is being explored. In this paper, the electrically conductive polymer--oxidized polypyrrole (PP)--has been evaluated for use as a substrate to enhance nerve cell interactions in culture as a first step toward potentially using such polymers to stimulate in vivo nerve regeneration. Image analysis demonstrates that PC-12 cells and primary chicken sciatic nerve explants attached and extended neurites equally well on both PP films and tissue culture polystyrene in the absence of electrical stimulation. In contrast, PC-12 cells interacted poorly with indium tin oxide (ITO), poly(L-lactic acid) (PLA), and poly(lactic acid-coglycolic acid) surfaces. However, PC-12 cells cultured on PP films and subjected to an electrical stimulus through the film showed a significant increase in neurite lengths compared with ones that were not subjected to electrical stimulation through the film and tissue culture polystyrene controls. The median neurite length for PC-12 cells grown on PP and subjected to an electrical stimulus was 18.14 μ m (n = 5643) compared with 9.5 μ m (n = 4440) for controls. Furthermore, animal implantation studies reveal that PP invokes little adverse tissue response compared with poly(lactic acid-coglycolic acid).

  10. 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...

  11. 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...

  12. Terpenoids with neurite outgrowth-promoting activity from the branches and leaves of Illicium merrillianum.

    PubMed

    Tian, Xin-Hui; Yue, Rong-Cai; Fang, Xin; Zhang, Jian-Ping; Wang, Guo-Wei; Shan, Lei; Zhang, Wei-Dong; Shen, Yun-Heng

    2016-05-01

    Eighteen terpenoids (1-18) were isolated from Illicium merrillianum. Compound 1 was identified as new compound, and its structure was established by comprehensive spectroscopic analysis and single-crystal X-ray diffraction. All compounds were evaluated for nerve growth factor (NGF)-mediated neurite outgrowth activity using rat pheochromocytoma (PC12) cells as a model system of neuronal differentiation. Compounds 1, 3, 18 showed significant neurite outgrowth-promoting activity in the presence of 20 ng/ml NGF in a dose-dependent manner at concentrations of 1-100 μM after 24-h treatment. Subtle difference of functional groups at C-2 position in hopane-type triterpene resulted in enormous bioactivity difference, compound 1 was neurotrophic but 2 was cytotoxic.

  13. An astrocytic binding site for neuronal Thy-1 and its effect on neurite outgrowth.

    PubMed Central

    Dreyer, E B; Leifer, D; Heng, J E; McConnell, J E; Gorla, M; Levin, L A; Barnstable, C J; Lipton, S A

    1995-01-01

    Thy-1, a member of the immunoglobulin superfamily, is one of the most abundant glycoproteins on mammalian neurons. Nevertheless, its role in the peripheral or central nervous system is poorly understood. Certain monoclonal antibodies to Thy-1 promote neurite outgrowth by rodent central nervous system neurons in vitro, suggesting that Thy-1 functions, in part, by modulating neurite outgrowth. We describe a binding site for Thy-1 on astrocytes. This Thy-1-binding protein has been characterized by immunofluroesence with specific anti-idiotype monoclonal antibodies and by three competitive binding assays using (i) anti-idiotype antibodies, (ii) purified Thy-1, and (iii) Thy-1-transfected cells. The Thy-1-binding protein may participate in axonal or dendritic development in the nervous system. Images Fig. 1 Fig. 3 PMID:7479964

  14. 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.

  15. Methylmercury decreases NGF-induced TrkA autophosphorylation and neurite outgrowth in PC12 cells.

    PubMed

    Parran, Damani K; Barone, Stanley; Mundy, William R

    2003-03-14

    Neurotrophin signaling through Trk receptors is important for differentiation and survival in the developing nervous system. The present study examined the effects of CH(3)Hg on (125)I-nerve growth factor (NGF) binding to the TrkA receptor, NGF-induced activation of the TrkA receptor, and neurite outgrowth in an in vitro model of differentiation using PC12 cells. Whole-cell binding assays using (125)I-NGF revealed a single binding site with a K(d) of approximately 1 nM. Methylmercury (CH(3)Hg) at 30 nM (EC(50) for neurite outgrowth inhibition) did not affect NGF binding to TrkA. TrkA autophosphorylation was measured by immunoblotting with a phospho-specific antibody. TrkA autophosphorylation peaked between 2.5 and 5 min of exposure and then decreased but was still detectable at 60 min. Concurrent exposure to CH(3)Hg and NGF for 2.5 min resulted in a concentration-dependent decrease in TrkA autophosphorylation, which was significant at 100 nM CH(3)Hg. To determine whether the observed inhibition of TrkA was sufficient to alter cell differentiation, NGF-stimulated neurite outgrowth was examined in PC12 cells after exposure to 30 nM CH(3)Hg, a concentration that inhibited TrkA autophosphorylation by approximately 50%. For comparison, a separate group of PC12 cells were exposed to a concentration of the selective Trk inhibitor K252a (30 nM), which had been shown to produce significant inhibition of TrkA autophosphorylation. Twenty-four hour exposure to either CH(3)Hg or K252a reduced neurite outgrowth to a similar degree. Our results suggest that CH(3)Hg may inhibit differentiation of PC12 cells by interfering with NGF-stimulated TrkA autophosphorylation.

  16. NRC-interacting factor directs neurite outgrowth in an activity-dependent manner.

    PubMed

    Zhao, X-S; Fu, W-Y; Hung, K-W; Chien, W W Y; Li, Z; Fu, A K; Ip, N Y

    2015-03-19

    Nuclear hormone receptor coregulator-interacting factor 1 (NIF-1) is a zinc finger nuclear protein that was initially identified to enhance nuclear hormone receptor transcription via its interaction with nuclear hormone receptor coregulator (NRC). NIF-1 may regulate gene transcription either by modulating general transcriptional machinery or remodeling chromatin structure through interactions with specific protein partners. We previously reported that the cytoplasmic/nuclear localization of NIF-1 is regulated by the neuronal Cdk5 activator p35, suggesting potential neuronal functions for NIF-1. The present study reveals that NIF-1 plays critical roles in regulating neuronal morphogenesis at early stages. NIF-1 was prominently expressed in the nuclei of developing rat cortical neurons. Knockdown of NIF-1 expression attenuated both neurite outgrowth in cultured cortical neurons and retinoic acid (RA)-treated Neuro-2a neuroblastoma cells. Furthermore, activity-induced Ca(2+) influx, which is critical for neuronal morphogenesis, stimulated the nuclear localization of NIF-1 in cortical neurons. Suppression of NIF-1 expression reduced the up-regulation of neuronal activity-dependent gene transcription. These findings collectively suggest that NIF-1 directs neuronal morphogenesis during early developmental stages through modulating activity-dependent gene transcription.

  17. Hydrocortisone Stimulates Neurite Outgrowth from Mouse Retinal Explants by Modulating Macroglial Activity

    PubMed Central

    Toops, Kimberly A.; Berlinicke, Cynthia; Zack, Donald J.; Nickells, Robert W.

    2012-01-01

    Purpose There is mounting evidence that retinal ganglion cells (RGCs) require a complex milieu of trophic factors to enhance cell survival and axon regeneration after optic nerve injury. The authors' goal was to examine the contribution of components of a combination of hormones, growth factors, steroids, and small molecules to creating a regenerative environment and to determine if any of these components modulated macroglial behavior to aid in regeneration. Methods Postnatal day 7 mouse retinal explants embedded in collagen were used as an in vitro model of neurite regeneration. Explants were treated with the culture supplements fetal bovine serum, N2, and G5 and a mixture of G5 and N2 components, designated enhanced N2 (EN2). Explants were evaluated for neurite outgrowth over 7 days in culture. The effects of each treatment were also evaluated on cultured RGCs purified by Thy1 immunopanning. Immunohistochemistry and qPCR analysis were used to evaluate differences in gene expression in the explants due to different treatments. Results EN2 stimulated significant neurite outgrowth from explants but not from purified RGCs. Elimination of hydrocortisone (HC) from EN2 reduced the mean neurites per explant by 37%. EN2-treated explants demonstrated increased expression of Gfap, Glul, Glt1, Cntf, Pedf, and VegfA compared with explants treated with EN2 without HC. Subsequent experiments showed that increased expression of Cntf and Glul was critical to the trophic effect of HC. Conclusions These data suggest that the HC in EN2 indirectly contributed to neurite outgrowth by activating macroglia to produce neurotrophic and neuroprotective molecules. PMID:22395888

  18. Role of Tetanus Neurotoxin Insensitive Vesicle-Associated Membrane Protein (Ti-Vamp) in Vesicular Transport Mediating Neurite Outgrowth

    PubMed Central

    Martinez-Arca, Sonia; Alberts, Philipp; Zahraoui, Ahmed; Louvard, Daniel; Galli, Thierry

    2000-01-01

    How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH2-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH2-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH2-terminal domain as a key regulator in this process. PMID:10811829

  19. SH2B1 orchestrates signaling events to filopodium formation during neurite outgrowth.

    PubMed

    Chen, Kuan-Wei; Chang, Yu-Jung; Chen, Linyi

    2015-01-01

    Morphogenesis during development is fundamental to the differentiation of several cell types. As neurite outgrowth marks neuritogenesis, formation of filopodia precede the formation of dendrites and axons. While the structure of filopodia is well-known, the initiation of filopodia during neurite outgrowth is not clear. SH2B1 is known to promote neurite outgrowth of PC12 cells, hippocampal and cortical neurons. As a signaling adaptor protein, SH2B1 interacts with several neurotrophin receptors, and regulates signaling as well as gene expression. Our recent findings suggest that SH2B1 can be recruited to the plasma membrane and F-actin fractions by IRSp53. IRSp53 bends plasma membrane and facilitates actin bundling to set the stage for filopodium formation. We further demonstrate that SH2B1-IRSp53 complexes enhance the formation of filopodia, dendrites and dendritic branches of hippocampal and cortical neurons. While the molecular mechanism underlying filopodium initiation is not clear, we propose that SH2B1-neurotrophin interacting sites may mark the putative sites of filopodium initiation.

  20. SH2B1 orchestrates signaling events to filopodium formation during neurite outgrowth

    PubMed Central

    Chen, Kuan-Wei; Chang, Yu-Jung; Chen, Linyi

    2015-01-01

    Morphogenesis during development is fundamental to the differentiation of several cell types. As neurite outgrowth marks neuritogenesis, formation of filopodia precede the formation of dendrites and axons. While the structure of filopodia is well-known, the initiation of filopodia during neurite outgrowth is not clear. SH2B1 is known to promote neurite outgrowth of PC12 cells, hippocampal and cortical neurons. As a signaling adaptor protein, SH2B1 interacts with several neurotrophin receptors, and regulates signaling as well as gene expression. Our recent findings suggest that SH2B1 can be recruited to the plasma membrane and F-actin fractions by IRSp53. IRSp53 bends plasma membrane and facilitates actin bundling to set the stage for filopodium formation. We further demonstrate that SH2B1-IRSp53 complexes enhance the formation of filopodia, dendrites and dendritic branches of hippocampal and cortical neurons. While the molecular mechanism underlying filopodium initiation is not clear, we propose that SH2B1-neurotrophin interacting sites may mark the putative sites of filopodium initiation. PMID:26479731

  1. 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

  2. Schwann cell migration and neurite outgrowth are influenced by media conditioned by epineurial fibroblasts.

    PubMed

    van Neerven, S G A; Pannaye, P; Bozkurt, A; Van Nieuwenhoven, F; Joosten, E; Hermans, E; Taccola, G; Deumens, R

    2013-11-12

    The regenerative capacity of the peripheral nervous system is largely related to Schwann cells undergoing proliferation and migration after injury and forming growth-supporting substrates for severed axons. Novel data show that fibroblasts to a certain extent regulate the pro-regenerative behavior of Schwann cells. In the setting of peripheral nerve injury, the fibroblasts that form the epineurium come into close contact with both Schwann cells and peripheral axons, but the potential influence on these latter two cell types has not been studied yet. In the present study we explored whether culture media, conditioned by epineurial fibroblasts can influence Schwann cells and/or neurite outgrowth from dorsal root ganglia neurons in vitro. Our data indicate that epineurial fibroblast-conditioned culture media substantially increase Schwann cell migration and the outgrowth of neurites. Schwann cell proliferation remained largely unaffected. These same read-out parameters were assayed in a condition where epineurial fibroblasts were subjected to stretch-cell-stress, a mechanical stressor that plays an important role in traumatic peripheral nerve injuries. Stretch-cell-stress of epineurial fibroblasts did not further change the positive effects of conditioned media on Schwann cell migration and neurite outgrowth. From these data we conclude that an as yet unknown pro-regenerative role can be attributed to epineurial fibroblasts, implying that such cells may affect the outcome of severe peripheral nerve injury. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. Gab1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells.

    PubMed

    Korhonen, J M; Saïd, F A; Wong, A J; Kaplan, D R

    1999-12-24

    The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.

  4. Secretin induces neurite outgrowth of PC12 through cAMP-mitogen-activated protein kinase pathway.

    PubMed

    Kim, Hyeon Soo; Yumkham, Sanatombi; Kim, Sun-Hee; Yea, Kyungmoo; Shin, You Chan; Ryu, Sung Ho; Suh, Pann-Ghill

    2006-02-28

    The gastrointestinal functions of secretin have been fairly well established. However, its function and mode of action within the nervous system remain largely unclear. To gain insight into this area, we have attempted to determine the effects of secretin on neuronal differentiation. Here, we report that secretin induces the generation of neurite outgrowth in pheochromocytoma PC12 cells. The expressions of Tau and beta-tubulin, neuronal differentiation markers, are increased upon secretin stimulation. In addition, secretin induces sustained mitogen-activated protein kinase (MAPK) activation and also stimulates the cAMP secretion. Moreover, the neurite outgrowth elicited by secretin is suppressed to a marked degree in the presence of either PD98059, a specific MAPK/ERK kinase (MEK) inhibitor, or H89, a specific protein kinase A (PKA) inhibitor. Taken together, these observations demonstrate that secretin induces neurite outgrowth of PC12 cells through cAMP- MAPK pathway, and provide a novel insight into the manner in which secretin participates in neuritogenesis.

  5. Oriented collagen as a potential cochlear implant electrode surface coating to achieve directed neurite outgrowth.

    PubMed

    Volkenstein, Stefan; Kirkwood, John E; Lai, Edwina; Dazert, Stefan; Fuller, Gerald G; Heller, Stefan

    2012-04-01

    In patients with severe to profound hearing loss, cochlear implants (CIs) are currently the only therapeutic option when the amplification with conventional hearing aids does no longer lead to a useful hearing experience. Despite its great success, there are patients in which benefit from these devices is rather limited. One reason may be a poor neuron-device interaction, where the electric fields generated by the electrode array excite a wide range of tonotopically organized spiral ganglion neurons at the cost of spatial resolution. Coating of CI electrodes to provide a welcoming environment combined with suitable surface chemistry (e.g. with neurotrophic factors) has been suggested to create a closer bioelectrical interface between the electrode array and the target tissue, which might lead to better spatial resolution, better frequency discrimination, and ultimately may improve speech perception in patients. Here we investigate the use of a collagen surface with a cholesteric banding structure, whose orientation can be systemically controlled as a guiding structure for neurite outgrowth. We demonstrate that spiral ganglion neurons survive on collagen-coated surfaces and display a directed neurite growth influenced by the direction of collagen fibril deposition. The majority of neurites grow parallel to the orientation direction of the collagen. We suggest collagen coating as a possible future option in CI technology to direct neurite outgrowth and improve hearing results for affected patients.

  6. Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats.

    PubMed

    Husmann, K; Faissner, A; Schachner, M

    1992-03-01

    The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats.

  7. Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats

    PubMed Central

    1992-01-01

    The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats. PMID:1371773

  8. Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro

    PubMed Central

    1995-01-01

    The amyloid precursor protein (APP) is a transmembrane protein expressed in several cell types. In the nervous system, APP is expressed by glial and neuronal cells, and several lines of evidence suggest that it plays a role in normal and pathological phenomena. To address the question of the actual function of APP in normal developing neurons, we undertook a study aimed at blocking APP expression using antisense oligonucleotides. Oligonucleotide internalization was achieved by linking them to a vector peptide that translocates through biological membranes. This original technique, which is very efficient and gives direct access to the cell cytosol and nucleus, allowed us to work with extracellular oligonucleotide concentrations between 40 and 200 nM. Internalization of antisense oligonucleotides overlapping the origin of translation resulted in a marked but transient decrease in APP neosynthesis that was not observed with the vector peptide alone, or with sense oligonucleotides. Although transient, the decrease in APP neosynthesis was sufficient to provoke a distinct decrease in axon and dendrite outgrowth by embryonic cortical neurons developing in vitro. The latter decrease was not accompanied by changes in the spreading of the cell bodies. A single exposure to coupled antisense oligonucleotides at the onset of the culture was sufficient to produce significant morphological effects 6, 18, and 24 h later, but by 42 h, there were no remaining significant morphologic changes. This report thus demonstrates that amyloid precursor protein plays an important function in the morphological differentiation of cortical neurons in primary culture. PMID:7876315

  9. Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels

    PubMed Central

    1993-01-01

    Electrical signals elicited by integrin interaction with ECM components and their role in neurite outgrowth were studied in two clones (N1 and N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitronectin (VN), this adhesion induced neurite outgrowth in N1 but not in N7 cells. Patch clamp recordings in whole cell configuration showed that, upon adhesion to FN or VN but not to platelet factor 4 (PF4), N1 cells undergo a marked (approximately equal to 20 mV) hyperpolarization of the resting potential (Vrest) that occurred within the first 20 min after cell contact with ECM, and persisted for approximately 1 h before reverting to the time zero values. This hyperpolarization was totally absent in N7 cells. A detailed analysis of the molecular mechanisms involved in N1 and N7 cell adhesion to ECM substrata was performed by using antibodies raised against the FN receptor and synthetic peptides variously competing with the FN or VN binding to integrin receptor (GRGDSP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N1 and N7 clones to FN and VN, revealing a similar implication of integrins in the adhesion of these clones to the ECM proteins. However, these anti-adhesive treatments, while ineffective on Vrest of N7 cells, abolished in N1 cells the FN- or VN-induced hyperpolarization and neurite outgrowth, that appeared therefore strictly associated and integrin-mediated phenomena. The nature of this association was deepened through a comparative analysis of the integrin profiles and the ion channels of N1 and N7 cells. The integrin immunoprecipitation profile resulted very similarly in the two clones, with only minor differences concerning the alpha V containing complexes. Both clones possessed Ca2+ and K+ delayed rectifier (KDR) channels, while only N1 cells were endowed with inward rectifier K+ (KIR) channels. The latter governed the Vrest, and, unlike KDR channels, were blocked by

  10. Signaling adaptor protein SH2B1 enhances neurite outgrowth and accelerates the maturation of human induced neurons.

    PubMed

    Hsu, Yi-Chao; Chen, Su-Liang; Wang, Ya-Jean; Chen, Yun-Hsiang; Wang, Dan-Yen; Chen, Linyi; Chen, Chia-Hsiang; Chen, Hwei-Hsien; Chiu, Ing-Ming

    2014-06-01

    Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.

  11. Signaling Adaptor Protein SH2B1 Enhances Neurite Outgrowth and Accelerates the Maturation of Human Induced Neurons

    PubMed Central

    Hsu, Yi-Chao; Chen, Su-Liang; Wang, Ya-Jean; Chen, Yun-Hsiang; Wang, Dan-Yen; Chen, Linyi; Chen, Chia-Hsiang; Chen, Hwei-Hsien

    2014-01-01

    Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling. PMID:24736401

  12. 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

  13. Binding of Cdc42 to phospholipase D1 is important in neurite outgrowth of neural stem cells

    SciTech Connect

    Yoon, Mee-Sup; Cho, Chan Ho; Lee, Ki Sung; Han, Joong-Soo . E-mail: jshan@hanyang.ac.kr

    2006-09-01

    We previously demonstrated that phospholipase D (PLD) expression and PLD activity are upregulated during neuronal differentiation. In the present study, employing neural stem cells from the brain cortex of E14 rat embryos, we investigated the role of Rho family GTPases in PLD activation and in neurite outgrowth of neural stem cells during differentiation. As neuronal differentiation progressed, the expression levels of Cdc42 and RhoA increased. Furthermore, Cdc42 and PLD1 were mainly localized in neurite, whereas RhoA was localized in cytosol. Co-immunoprecipitation revealed that Cdc42 was bound to PLD1 during differentiation, whereas RhoA was associated with PLD1 during both proliferation and differentiation. These results indicate that the association between Cdc42 and PLD1 is related to neuronal differentiation. To examine the effect of Cdc42 on PLD activation and neurite outgrowth, we transfected dominant negative Cdc42 (Cdc42N17) and constitutively active Cdc42 (Cdc42V12) into neural stem cells, respectively. Overexpression of Cdc42N17 decreased both PLD activity and neurite outgrowth, whereas co-transfection with Cdc42N17 and PLD1 restored them. On the other hand, Cdc42V12 increased both PLD activity and neurite outgrowth, suggesting that active state of Cdc42 is important in upregulation of PLD activity which is responsible for the increase of neurite outgrowth.

  14. Non-cytotoxic Concentration of Cisplatin Decreases Neuroplasticity-Related Proteins and Neurite Outgrowth Without Affecting the Expression of NGF in PC12 Cells.

    PubMed

    Ferreira, Rafaela Scalco; Dos Santos, Neife Aparecida Guinaim; Martins, Nádia Maria; Fernandes, Laís Silva; Dos Santos, Antonio Cardozo

    2016-11-01

    Cisplatin is the most effective and neurotoxic platinum chemotherapeutic agent. It induces a peripheral neuropathy characterized by distal axonal degeneration that might progress to degeneration of cell bodies and apoptosis. Most symptoms occur nearby distal axonal branches and axonal degeneration might induce peripheral neuropathy regardless neuronal apoptosis. The toxic mechanism of cisplatin has been mainly associated with DNA damage, but cisplatin might also affect neurite outgrowth. Nevertheless, the neurotoxic mechanism of cisplatin remains unclear. We investigated the early effects of cisplatin on axonal plasticity by using non-cytotoxic concentrations of cisplatin and PC12 cells as a model of neurite outgrowth and differentiation. PC12 cells express NGF-receptors (trkA) and respond to NGF by forming neurites, branches and synaptic vesicles. For comparison, we used a neuronal model (SH-SY5Y cells) that does not express trkA nor responds to NGF. Cisplatin did not change NGF expression in PC12 cells and decreased neurite outgrowth in both models, suggesting a NGF/trkA independent mechanism. It also reduced axonal growth (GAP-43) and synaptic (synapsin I and synaptophysin) proteins in PC12 cells, without inducing mitochondrial damage or apoptosis. Therefore, cisplatin might affect axonal plasticity before DNA damage, NGF/trkA down-regulation, mitochondrial damage or neuronal apoptosis. This is the first study to show that neuroplasticity-related proteins might be early targets of the neurotoxic action of cisplatin and their role on cisplatin-induced peripheral neuropathy should be investigated in vivo.

  15. Ca2+ transients are not required as signals for long-term neurite outgrowth from cultured sympathetic neurons

    PubMed Central

    1990-01-01

    A method for clamping cytosolic free Ca2+ ([Ca2+]i) in cultures of rat sympathetic neurons at or below resting levels for several days was devised to determine whether Ca2+ signals are required for neurite outgrowth from neurons that depend on Nerve Growth Factor (NGF) for their growth and survival. To control [Ca2+]i, normal Ca2+ influx was eliminated by titration of extracellular Ca2+ with EGTA and reinstated through voltage-sensitive Ca2+ channels. The rate of neurite outgrowth and the number of neurites thus became dependent on the extent of depolarization by KCl, and withdrawal of KCl caused an immediate cessation of growth. Neurite outgrowth was completely blocked by the L type Ca2+ channel antagonists nifedipine, nitrendipine, D600, or diltiazem at sub- or micromolar concentrations. Measurement of [Ca2+]i in cell bodies using the fluorescent Ca2+ indicator fura-2 established that optimal growth, similar to that seen in normal medium, was obtained when [Ca2+]i was clamped at resting levels. These levels of [Ca2+]i were set by serum, which elevated [Ca2+]i by integral of 30 nM, whereas the addition of NGF had no effect on [Ca2+]i. The reduction of [Ca2+]o prevented neurite fasciculation but this had no effect on the rate of neurite elongation or on the number of extending neurites. These results show that neurite outgrowth from NGF-dependent neurons occurs over long periods in the complete absence of Ca2+ signals, suggesting that Ca2+ signals are not necessary for operating the basic machinery of neurite outgrowth. PMID:2324199

  16. Liraglutide Promotes Cortical Neurite Outgrowth via the MEK-ERK Pathway.

    PubMed

    Li, Meng; Li, Shilun; Li, Yukun

    2015-10-01

    Liraglutide is the glucagon-like peptide-1 (GLP-1) synthetic form which has been approved by the US Food and Drug Administration to be released onto the market. The metabolic benefits of incretin hormone as an anti-diabetic agent are widely recognized, but its potential extra-pancreatic effects of GLP-1 analog (liraglutide) in the central nerve system are less well known. To this purpose, we used immunofluorescence method to examine the effect of liraglutide on neurite outgrowth in primary cortical neuron culture by measuring neurite length and confirmed the promotion effect. Then, we investigated the potential mechanisms and found that liraglutide promoted neurite outgrowth in a dose-dependant manner, and this effect could be partially inhibited by MEK-ERK inhibitor U0126. Besides, liraglutide induced an increase of p-ERK/ERK expression, which could be blocked in the presence of U0126. Similarly, phosphorylated transcription factor (p-CREB) level shared the same trend with p-ERK/ERK ratio after liraglutide treatment. Collectively, our data illustrated that that liraglutide exerts neurotrophin-like activity partly via MEK-ERK pathway, which might offer a novel idea for treatment of axon-associated neurological diseases.

  17. RA-RAR-β counteracts myelin-dependent inhibition of neurite outgrowth via Lingo-1 repression.

    PubMed

    Puttagunta, Radhika; Schmandke, André; Floriddia, Elisa; Gaub, Perrine; Fomin, Natalie; Ghyselinck, Norbert B; Di Giovanni, Simone

    2011-06-27

    After an acute central nervous system injury, axonal regeneration is limited as the result of a lack of neuronal intrinsic competence and the presence of extrinsic inhibitory signals. The injury fragments the myelin neuronal insulating layer, releasing extrinsic inhibitory molecules to signal through the neuronal membrane-bound Nogo receptor (NgR) complex. In this paper, we show that a neuronal transcriptional pathway can interfere with extrinsic inhibitory myelin-dependent signaling, thereby promoting neurite outgrowth. Specifically, retinoic acid (RA), acting through the RA receptor β (RAR-β), inhibited myelin-activated NgR signaling through the transcriptional repression of the NgR complex member Lingo-1. We show that suppression of Lingo-1 was required for RA-RAR-β to counteract extrinsic inhibition of neurite outgrowth. Furthermore, we confirm in vivo that RA treatment after a dorsal column overhemisection injury inhibited Lingo-1 expression, specifically through RAR-β. Our findings identify a novel link between RA-RAR-β-dependent proaxonal outgrowth and inhibitory NgR complex-dependent signaling, potentially allowing for the development of molecular strategies to enhance axonal regeneration after a central nervous system injury.

  18. GSK-3β activation mediates Nogo-66-induced inhibition of neurite outgrowth in N2a cells.

    PubMed

    Shen, Jian-ying; Yi, Xu-xia; Xiong, Nan-xiang; Wang, Hai-jun; Duan, Xiao-wei; Zhao, Hong-yang

    2011-11-14

    The axons of the adult mammalian brain and spinal cord fail to regenerate after injury, and it has been suggested that Nogo-66 could prevent CNS axon repair. However, the mechanism of Nogo-66 inhibiting neurite outgrowth remains unknown. Our previous results indicated that protein kinase B (PKB) is involved in the inhibition of the neurite outgrowth by Nogo-66. Glycogen synthase kinase-3β (GSK-3β) is implicated in many processes in the nervous system, including differentiation, specification, polarity, plasticity and axon growth. In addition, GSK-3β is one of the most important molecules downstream of PKB. In the present study, we report on the role of GSK-3β signaling on Nogo-66-treated mouse neuroblastoma N2a cells. Nogo-66 reduced the phosphorylation of GSK-3β at Ser9 in N2a cells. In contrast, pretreatment with SB216763, a specific inhibitor of GSK-3β, resulted in an amelioration of neurite outgrowth by Nogo-66, compared with the Nogo-66 alone group (P<0.05). Moreover, we performed RNA interference experiments to knock down GSK-3β expression levels in N2a cells via transient transfection of shRNA plasmids. The inhibition of neurite outgrowth by Nogo-66 was subdued in shRNA cells, compared to the non-RNAi cells (P<0.05). Taken together, these data suggest that GSK-3β is involved in the inhibition by Nogo-66 of neurite outgrowth in N2a cells.

  19. μ2-Dependent endocytosis of N-cadherin is regulated by β-catenin to facilitate neurite outgrowth.

    PubMed

    Chen, Yi-Ting; Tai, Chin-Yin

    2017-02-22

    Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N-cadherin, a calcium-dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N-cadherin internalizes through clathrin-mediated endocytosis (CME). Two tyrosine-based motifs in the cytoplasmic domain of N-cadherin recognized by the μ2 subunit of the AP-2 adaptor complex are responsible for CME of N-cadherin. Moreover, β-catenin, a core component of the N-cadherin adhesion complex, inhibits N-cadherin endocytosis by masking the 2 tyrosine-based motifs. Removal of β-catenin facilitates μ2 binding to N-cadherin, thereby increasing clathrin-mediated N-cadherin endocytosis and neurite outgrowth without affecting the steady-state level of surface N-cadherin. These results identify and characterize the mechanism controlling N-cadherin endocytosis through β-catenin-regulated μ2 binding to modulate neurite outgrowth.

  20. Exposure to 1800 MHz radiofrequency radiation impairs neurite outgrowth of embryonic neural stem cells

    PubMed Central

    Chen, Chunhai; Ma, Qinlong; Liu, Chuan; Deng, Ping; Zhu, Gang; Zhang, Lei; He, Mindi; Lu, Yonghui; Duan, Weixia; Pei, Liping; Li, Min; Yu, Zhengping; Zhou, Zhou

    2014-01-01

    A radiofrequency electromagnetic field (RF-EMF) of 1800 MHz is widely used in mobile communications. However, the effects of RF-EMFs on cell biology are unclear. Embryonic neural stem cells (eNSCs) play a critical role in brain development. Thus, detecting the effects of RF-EMF on eNSCs is important for exploring the effects of RF-EMF on brain development. Here, we exposed eNSCs to 1800 MHz RF-EMF at specific absorption rate (SAR) values of 1, 2, and 4 W/kg for 1, 2, and 3 days. We found that 1800 MHz RF-EMF exposure did not influence eNSC apoptosis, proliferation, cell cycle or the mRNA expressions of related genes. RF-EMF exposure also did not alter the ratio of eNSC differentiated neurons and astrocytes. However, neurite outgrowth of eNSC differentiated neurons was inhibited after 4 W/kg RF-EMF exposure for 3 days. Additionally, the mRNA and protein expression of the proneural genes Ngn1 and NeuroD, which are crucial for neurite outgrowth, were decreased after RF-EMF exposure. The expression of their inhibitor Hes1 was upregulated by RF-EMF exposure. These results together suggested that 1800 MHz RF-EMF exposure impairs neurite outgrowth of eNSCs. More attention should be given to the potential adverse effects of RF-EMF exposure on brain development. PMID:24869783

  1. 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.

  2. Tenascin-C contains distinct adhesive, anti-adhesive, and neurite outgrowth promoting sites for neurons

    PubMed Central

    1996-01-01

    The glia-derived extracellular matrix glycoprotein tenascin-C (TN-C) is transiently expressed in the developing CNS and may mediate neuron-glia interactions. Perturbation experiments with specific monoclonal antibodies suggested that TN-C functions for neural cells are encoded by distinct sites of the glycoprotein (Faissner, A., A. Scholze, and B. Gotz. 1994. Tenascin glycoproteins in developing neural tissues--only decoration? Persp. Dev. Neurobiol. 2:53-66). To characterize these further, bacterially expressed recombinant domains were generated and used for functional studies. Several short-term-binding sites for mouse CNS neurons could be assigned to the fibronectin type III (FNIII) domains. Of these, the alternatively spliced insert TNfnA1,2,4,B,D supported initial attachment for both embryonic day 18 (E18) rat and postnatal day 6 (P6) mouse neurons. Only TNfn1-3 supported binding and growth of P6 mouse cerebellar neurons after 24 h, whereas attachment to the other domains proved reversible and resulted in cell detachment or aggregation. In choice assays on patterned substrates, repulsive properties could be attributed to the EGF-type repeats TNegf, and to TNfnA1,2,4. Finally, neurite outgrowth promoting properties for E18 rat hippocampal neurons and P0 mouse DRG explants could be assigned to TNfnB,D, TNfnD,6, and TNfn6. The epitope of mAb J1/tn2 which abolishes the neurite outgrowth inducing effect of intact TN-C could be allocated to TNfnD. These observations suggest that TN-C harbors distinct cell- binding, repulsive, and neurite outgrowth promoting sites for neurons. Furthermore, the properties of isoform-specific TN-C domains suggest functional significance of the alternative splicing of TN-C glycoproteins. PMID:8647898

  3. Exposure to 1800 MHz radiofrequency radiation impairs neurite outgrowth of embryonic neural stem cells.

    PubMed

    Chen, Chunhai; Ma, Qinlong; Liu, Chuan; Deng, Ping; Zhu, Gang; Zhang, Lei; He, Mindi; Lu, Yonghui; Duan, Weixia; Pei, Liping; Li, Min; Yu, Zhengping; Zhou, Zhou

    2014-05-29

    A radiofrequency electromagnetic field (RF-EMF) of 1800 MHz is widely used in mobile communications. However, the effects of RF-EMFs on cell biology are unclear. Embryonic neural stem cells (eNSCs) play a critical role in brain development. Thus, detecting the effects of RF-EMF on eNSCs is important for exploring the effects of RF-EMF on brain development. Here, we exposed eNSCs to 1800 MHz RF-EMF at specific absorption rate (SAR) values of 1, 2, and 4 W/kg for 1, 2, and 3 days. We found that 1800 MHz RF-EMF exposure did not influence eNSC apoptosis, proliferation, cell cycle or the mRNA expressions of related genes. RF-EMF exposure also did not alter the ratio of eNSC differentiated neurons and astrocytes. However, neurite outgrowth of eNSC differentiated neurons was inhibited after 4 W/kg RF-EMF exposure for 3 days. Additionally, the mRNA and protein expression of the proneural genes Ngn1 and NeuroD, which are crucial for neurite outgrowth, were decreased after RF-EMF exposure. The expression of their inhibitor Hes1 was upregulated by RF-EMF exposure. These results together suggested that 1800 MHz RF-EMF exposure impairs neurite outgrowth of eNSCs. More attention should be given to the potential adverse effects of RF-EMF exposure on brain development.

  4. Screening of natural medicines that efficiently activate neurite outgrowth in PC12 cells in C2C12-cultured medium.

    PubMed

    Uezato, Tadayoshi; Sato, Eiji; Miura, Naoyuki

    2012-02-01

    We have studied the effects of natural medicines on neurite outgrowth in PC12D cells in a cultured medium of C2C12 cells. Derived from mouse myoblasts, the C2C12 cells secrete neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). The secretion of these neurotrophins from C2C12 cells stimulate neurite outgrowth in PC12D cells. We have screened a total of 120 samples and found five natural medicines: Trichosanthes Root, Asiasarum Root, Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex, that enhance the activity of C2C12-cultured medium to stimulate neurite outgrowth in PC12D cells. These natural medicines promoted not only neurite outgrowth but also stabilized the neurite formation in PC12D cells for several days. RT-PCR analysis showed that NGF was significantly increased with Trichosanthes and Lycium Bark. However, BDNF was slightly decreased with Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex. NT-3 was increased slightly by all of these natural medicines except Sinomenium Stem. All these five natural medicines significantly increased the number and length of neurites in PC12D cells in co-culture with C2C12 cells.

  5. 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

  6. Topiramate promotes neurite outgrowth and recovery of function after nerve injury.

    PubMed

    Smith-Swintosky, V L; Zhao, B; Shank, R P; Plata-Salaman, C R

    2001-04-17

    Topiramate is a structurally novel neurotherapeutic agent with a unique combination of pharmacological properties and currently is available in most world markets for treating several seizure disorders. Because its pharmacological profile was suggestive of possible activity as a neuroprotectant, topiramate was evaluated and found to be active in several animal models of stroke or neuropathic pain. This prompted an evaluation of topiramate as a possible neurotrophic agent. In this study, topiramate enhanced the recovery of facial nerve function after injury when administered orally at therapeutically relevant doses, and significantly increased neurite outgrowth in cell cultures derived from fetal rat cortical and hippocampal tissues.

  7. PAK–PIX interactions regulate adhesion dynamics and membrane protrusion to control neurite outgrowth

    PubMed Central

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

    2013-01-01

    Summary 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

  8. 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.

  9. 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

  10. Dual Regulation of RA-RhoGAP Activity by Phosphatidic Acid and Rap1 during Neurite Outgrowth*

    PubMed Central

    Kurooka, Takao; Yamamoto, Yasunori; Takai, Yoshimi; Sakisaka, Toshiaki

    2011-01-01

    During neurite outgrowth, Rho small G protein activity is spatiotemporally regulated to organize the neurite sprouting, extension, and branching. We have previously identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 small G protein in the neurite outgrowth. In addition to the Ras-associating (RA) domain for Rap1 binding, RA-RhoGAP has the pleckstrin homology (PH) domain for lipid binding. Here, we showed that phosphatidic acid (PA) bound to the PH domain and enhanced GAP activity for Rho. RA-RhoGAP induced extension of neurite in a diacylglycerol kinase-mediated synthesis of the PA-dependent manner. Knockdown of RA-RhoGAP reduced the diacylglycerol kinase-induced neurite extension. In contrast to the effect of the RA domain, the PH domain was specifically involved in the neurite extension, not in the sprouting and branching. These results indicate that PA and Rap1 cooperatively regulate RA-RhoGAP activity for promoting neurite outgrowth. PMID:21169361

  11. Synergistic Effects of 3D ECM and Chemogradients on Neurite Outgrowth and Guidance: A Simple Modeling and Microfluidic Framework

    PubMed Central

    Srinivasan, Parthasarathy; Zervantonakis, Ioannis K.; Kothapalli, Chandrasekhar R.

    2014-01-01

    During nervous system development, numerous cues within the extracellular matrix microenvironment (ECM) guide the growing neurites along specific pathways to reach their intended targets. Neurite motility is controlled by extracellular signal sensing through the growth cone at the neurite tip, including chemoattractive and repulsive cues. However, it is difficult to regenerate and restore neurite tracts, lost or degraded due to an injury or disease, in the adult central nervous system. Thus, it is important to evaluate the dynamic interplay between ECM and the concentration gradients of these cues, which would elicit robust neuritogenesis. Such information is critical in understanding the processes involved in developmental biology, and in developing high-fidelity neurite regenerative strategies post-injury, and in drug discovery and targeted therapeutics for neurodegenerative conditions. Here, we quantitatively investigated this relationship using a combination of mathematical modeling and in vitro experiments, and determined the synergistic role of guidance cues and ECM on neurite outgrowth and turning. Using a biomimetic microfluidic system, we have shown that cortical neurite outgrowth and turning under chemogradients (IGF-1 or BDNF) within 3D scaffolds is highly regulated by the source concentration of the guidance cue and the physical characteristics of the scaffold. A mechanistic-driven partial differential equation model of neurite outgrowth has been proposed, which could also be used prospectively as a predictive tool. The parameters for the chemotaxis term in the model are determined from the experimental data using our microfluidic assay. Resulting model simulations demonstrate how neurite outgrowth was critically influenced by the experimental variables, which was further supported by experimental data on cell-surface-receptor expressions. The model results are in excellent agreement with the experimental findings. This integrated approach represents a

  12. A New Role for TIMP-1 in Modulating Neurite Outgrowth and Morphology of Cortical Neurons

    PubMed Central

    Ould-yahoui, Adlane; Tremblay, Evelyne; Sbai, Oualid; Ferhat, Lotfi; Bernard, Anne; Charrat, Eliane; Gueye, Yatma; Lim, Ngee Han; Brew, Keith; Risso, Jean-Jacques; Dive, Vincent; Khrestchatisky, Michel; Rivera, Santiago

    2009-01-01

    Background Tissue inhibitor of metalloproteinases-1 (TIMP-1) displays pleiotropic activities, both dependent and independent of its inhibitory activity on matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMP-1 is strongly upregulated in reactive astrocytes and cortical neurons following excitotoxic/inflammatory stimuli, but no information exists on its effects on growth and morphology of cortical neurons. Principal Findings We found that 24 h incubation with recombinant TIMP-1 induced a 35% reduction in neurite length and significantly increased growth cones size and the number of F-actin rich microprocesses. TIMP-1 mediated reduction in neurite length affected both dendrites and axons after 48 h treatment. The effects on neurite length and morphology were not elicited by a mutated form of TIMP-1 inactive against MMP-1, -2 and -3, and still inhibitory for MMP-9, but were mimicked by a broad spectrum MMP inhibitor. MMP-9 was poorly expressed in developing cortical neurons, unlike MMP-2 which was present in growth cones and whose selective inhibition caused neurite length reductions similar to those induced by TIMP-1. Moreover, TIMP-1 mediated changes in cytoskeleton reorganisation were not accompanied by modifications in the expression levels of actin, βIII-tubulin, or microtubule assembly regulatory protein MAP2c. Transfection-mediated overexpression of TIMP-1 dramatically reduced neuritic arbour extension in the absence of detectable levels of released extracellular TIMP-1. Conclusions Altogether, TIMP-1 emerges as a modulator of neuronal outgrowth and morphology in a paracrine and autrocrine manner through the inhibition, at least in part, of MMP-2 and not MMP-9. These findings may help us understand the role of the MMP/TIMP system in post-lesion pre-scarring conditions. PMID:20011518

  13. 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. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  14. Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin.

    PubMed Central

    Müller, U; Bossy, B; Venstrom, K; Reichardt, L F

    1995-01-01

    The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin. Images PMID:7626807

  15. Spatial gene's (Tbata) implication in neurite outgrowth and dendrite patterning in hippocampal neurons.

    PubMed

    Yammine, Miriam; Saade, Murielle; Chauvet, Sophie; Nguyen, Catherine

    2014-03-01

    The unique architecture of neurons requires the establishment and maintenance of polarity, which relies in part on microtubule-based kinesin motor transport to deliver essential cargo into axons and dendrites. In developing neurons, kinesin trafficking is essential for delivering organelles and molecules that are crucial for elongation and guidance of the growing axonal and dendritic termini. In mature neurons, kinesin cargo delivery is essential for neuron dynamic physiological functions which are critical in brain development. In this work, we followed Spatial (Tbata) gene expression during primary hippocampal neuron development and showed that it is highly expressed during dendrite formation. Spatial protein exhibits a somatodendritic distribution and we show that the kinesin motor Kif17, among other dendrite specific kinesins, is crucial for Spatial localization to dendrites of hippocampal neurons. Furthermore, Spatial down regulation in primary hippocampal cells revealed a role for Spatial in maintaining neurons' polarity by ensuring proper neurite outgrowth. This polarity is specified by intrinsic and extracellular signals that allow neurons to determine axon and dendrite fate during development. Neurotrophic factors, such as the Nerve Growth Factor (NGF), are candidate extracellular polarity-regulating cues which are proposed to accelerate neuronal polarization by enhancing dendrite growth. Here, we show that NGF treatment increases Spatial expression in hippocampal neurons. Altogether, these data suggest that Spatial, in response to NGF and through its transport by Kif17, is crucial for neuronal polarization and can be a key regulator of neurite outgrowth.

  16. Endoplasmic Reticulum-Localized Transmembrane Protein Dpy19L1 Is Required for Neurite Outgrowth

    PubMed Central

    Watanabe, Keisuke; Bizen, Norihisa; Sato, Noboru; Takebayashi, Hirohide

    2016-01-01

    The endoplasmic reticulum (ER), including the nuclear envelope, is a continuous and intricate membrane-bound organelle responsible for various cellular functions. In neurons, the ER network is found in cell bodies, axons, and dendrites. Recent studies indicate the involvement of the ER network in neuronal development, such as neuronal migration and axonal outgrowth. However, the regulation of neural development by ER-localized proteins is not fully understood. We previously reported that the multi-transmembrane protein Dpy19L1 is required for neuronal migration in the developing mouse cerebral cortex. A Dpy19L family member, Dpy19L2, which is a causative gene for human Globozoospermia, is suggested to act as an anchor of the acrosome to the nuclear envelope. In this study, we found that the patterns of exogenous Dpy19L1 were partially coincident with the ER, including the nuclear envelope in COS-7 cells at the level of the light microscope. The reticular distribution of Dpy19L1 was disrupted by microtubule depolymerization that induces retraction of the ER. Furthermore, Dpy19L1 showed a similar distribution pattern with a ER marker protein in embryonic mouse cortical neurons. Finally, we showed that Dpy19L1 knockdown mediated by siRNA resulted in decreased neurite outgrowth in cultured neurons. These results indicate that transmembrane protein Dpy19L1 is localized to the ER membrane and regulates neurite extension during development. PMID:27959946

  17. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds.

    PubMed

    Holmes, T C; de Lacalle, S; Su, X; Liu, G; Rich, A; Zhang, S

    2000-06-06

    A new type of self-assembling peptide (sapeptide) scaffolds that serve as substrates for neurite outgrowth and synapse formation is described. These peptide-based scaffolds are amenable to molecular design by using chemical or biotechnological syntheses. They can be tailored to a variety of applications. The sapeptide scaffolds are formed through the spontaneous assembly of ionic self-complementary beta-sheet oligopeptides under physiological conditions, producing a hydrogel material. The scaffolds can support neuronal cell attachment and differentiation as well as extensive neurite outgrowth. Furthermore, they are permissive substrates for functional synapse formation between the attached neurons. That primary rat neurons form active synapses on such scaffold surfaces in situ suggests these scaffolds could be useful for tissue engineering applications. The buoyant sapeptide scaffolds with attached cells in culture can be transported readily from one environment to another. Furthermore, these peptides did not elicit a measurable immune response or tissue inflammation when introduced into animals. These biological materials created through molecular design and self assembly may be developed as a biologically compatible scaffold for tissue repair and tissue engineering.

  18. Plexin B3 promotes neurite outgrowth, interacts homophilically, and interacts with Rin

    PubMed Central

    Hartwig, Christine; Veske, Andres; Krejcova, Sarka; Rosenberger, Georg; Finckh, Ulrich

    2005-01-01

    Background Plexins, known to date as receptors of semaphorins, are implicated in semaphorin-mediated axon repulsion and growth cone collapse. However, subtype-specific functions of the majority of the nine members of the mammalian plexin family are largely unknown. In order to investigate functional properties of B-plexins, we analyzed the expression of human and murine plexin B3 and expressed full-length human plexins B2 (B2) and B3 (B3) in NIH-3T3 cells. Results Unexpectedly, B3 strongly and B2 moderately stimulate neurite outgrowth of primary murine cerebellar neurons. Both plexins mediate Ca2+/Mg2+-dependent cell aggregation due to homophilic trans-interaction, which is strong in the case of B3 and moderate for B2. Using different deletion constructs we show that the sema domain of B3 is essential for homophilic interaction. Using yeast two-hybrid analysis, we identified the neuron-specific and calmodulin-binding Ras-related GTPase Rin as an interaction partner of the intracellular part of B3, but not of B2. Rin, also known for its neurite outgrowth-inducing characteristics, co-localizes and co-immunoprecipitates with B3 in co-transfected COS-7 cells. Conclusion Our data suggest an involvement of homophilic interaction of B3 in semaphorin-independent signaling mechanisms positively influencing neuronal morphogenesis or function. Furthermore the neuron-specific small GTPase Rin is involved in downstream signaling of plexin B3. PMID:16122393

  19. Atlastin regulates store-operated calcium entry for nerve growth factor-induced neurite outgrowth

    PubMed Central

    Li, Jing; Yan, Bing; Si, Hongjiang; Peng, Xu; Zhang, Shenyuan L.; Hu, Junjie

    2017-01-01

    Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by a class of dynamin-like GTPases known as atlastin (ATL). Depletion of or mutations in ATL cause an unbranched ER morphology and hereditary spastic paraplegia (HSP), a neurodegenerative disease characterized by axon shortening in corticospinal motor neurons and progressive spasticity of the lower limbs. How ER shaping is linked to neuronal defects is poorly understood. Here, we show that dominant-negative mutants of ATL1 in PC-12 cells inhibit nerve growth factor (NGF)-induced neurite outgrowth. Overexpression of wild-type or mutant ATL1 or depletion of ATLs alters ER morphology and affects store-operated calcium entry (SOCE) by decreasing STIM1 puncta formation near the plasma membrane upon calcium depletion of the ER. In addition, blockage of the STIM1-Orai pathway effectively abolishes neurite outgrowth of PC-12 cells stimulated by NGF. These results suggest that SOCE plays an important role in neuronal regeneration, and mutations in ATL1 may cause HSP, partly by undermining SOCE. PMID:28240257

  20. 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.

  1. Enhanced neural cell adhesion and neurite outgrowth on graphene-based biomimetic substrates.

    PubMed

    Hong, Suck Won; Lee, Jong Ho; Kang, Seok Hee; Hwang, Eun Young; Hwang, Yu-Shik; Lee, Mi Hee; Han, Dong-Wook; 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.

  2. 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

  3. A patterned recombinant human IgM guides neurite outgrowth of CNS neurons.

    PubMed

    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.

  4. 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

  5. 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.

  6. SCG10, a microtubule destabilizing factor, stimulates the neurite outgrowth by modulating microtubule dynamics in rat hippocampal primary cultured neurons.

    PubMed

    Morii, Hiroshi; Shiraishi-Yamaguchi, Yoko; Mori, Nozomu

    2006-09-01

    Microtubule dynamics, one of the key elements in neurite outgrowth, is regulated by various regulatory factors to determine the behavior of the neuronal growth cone and to form the specialized neuronal shape. SCG10 is a neuron-specific stathmin protein with a potent microtubule destabilizing factor and is enriched in the growth cones of the developing neurons. We investigated the functional role of SCG10 in neurite outgrowth using rat hippocampal primary cultured neurons. Genetic manipulation of SCG10 using a short-interfering RNA duplex markedly decreased the SCG10 expression level and significantly suppressed neurite outgrowth. This result was confirmed by immunodepletion experiments. On the other hand, the protein transduction of SCG10 using a polyarginine tag stimulated neurite outgrowth. Such manipulation of the SCG10 expression level affected microtubule morphology within the growth cones. A decrease in the SCG10 level converted the morphology to a more stable state, while an increase converted the morphology to a more dynamic state. However, an excess of SCG10 induced neurite retraction due to an excess of microtubule disassembly. These results suggest that SCG10 serves as an important regulatory factor of growth cone motility by enhancing microtubule dynamics, possibly through increasing the catastrophe frequency.

  7. 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

  8. Potential Mechanism of Neurite Outgrowth Enhanced by Electrical Stimulation: Involvement of MicroRNA-363-5p Targeting DCLK1 Expression in Rat.

    PubMed

    Quan, Xin; Huang, Liangliang; Yang, Yafeng; Ma, Teng; Liu, Zhongyang; Ge, Jun; Huang, Jinghui; Luo, Zhuojing

    2017-02-01

    Electrical stimulation (ES) promotes neurite outgrowth and nerve regeneration, but the underlying mechanisms remain undefined. In the present study, we investigated the role of micro RNAs (miRNAs) in ES-mediated neurite outgrowth. First, we performed microarray analyses to identify changes in the miRNAs profile of dorsal root ganglion neurons (DRGNs) following ES. The expression of 16 known miRNAs was altered by ES. Bioinformatics showed that the potential targets of these differentially expressed miRNAs were involved in neurite outgrowth. We focused on miRNA-363-5p (miR-363-5p), because its expression was consistently altered by ES in the present study. Silencing miR-363-5p promoted neurite outgrowth, while miR-363-5p mimic reduced neurite outgrowth. Downregulation of miR-363-5p indicated that double cortin-like kinase (DCLK) 1, a major microtubule-associated protein, was a direct target of miR-363-5p in DRGNs. Knockdown of DCLK1 recapitulated the beneficial effect of a miR-363-5p inhibitor on DRG neurite outgrowth. In conclusion, our data has indicated that miR-363-5p is involved in ES-promoted neurite outgrowth by targeting DCLK1. These findings provide new insights into the roles of miRNAs in ES-enhanced neurite outgrowth and regeneration.

  9. Outgrowth of Neurites from NIE-115 Neuroblastoma Cells Is Prevented on Repulsive Substrates through the Action of PAK

    PubMed Central

    Marler, Katharine J. M.; Kozma, Robert; Ahmed, Sohail; Dong, Jing-Ming; Hall, Christine; Lim, Louis

    2005-01-01

    In the central nervous system (CNS), damaged axons are inhibited from regeneration by glial scars, where secreted chondroitin sulfate proteoglycan (CSPG) and tenascin repulse outgrowth of neurites, the forerunners of axons and dendrites. During differentiation, these molecules are thought to form boundaries for guiding neurons to their correct targets. In neuroblastoma NIE-115 cells, outgrowth of neurites on laminin could be induced by serum starvation or inhibition of RhoA by Clostridium botulinum C3 toxin. The outgrowing neurites avoided crossing onto the repulsive substrate CSPG or tenascin. This avoidance response was partially overcome on expression of membrane-targeted and kinase-inactive forms of PAK. In these cells, the endogenous PAK isoforms colocalized with actin in distinctive sites, αPAK in the cell center as small clusters and along the neurite shaft and βPAK and γPAK in areas with membrane ruffles and filopodia, respectively. When isoform-specific N-terminal PAK sequences were introduced to interfere with PAK function, substantially more neurites crossed onto CSPG when cells contained a γPAK-derived peptide but not the corresponding αPAK- or βPAK-derived peptide. Thus, while neurite outgrowth can be promoted by RhoA inhibition, overcoming the accompanying repulsive guidance response will require modulation of PAK activity. These results have therapeutic implications for CNS repair processes. PMID:15923637

  10. Integrin α5β1 expression on dopaminergic neurons is involved in dopaminergic neurite outgrowth on striatal neurons

    PubMed Central

    Izumi, Yasuhiko; Wakita, Seiko; Kanbara, Chisato; Nakai, Toshie; Akaike, Akinori; Kume, Toshiaki

    2017-01-01

    During development, dopaminergic neurons born in the substantia nigra extend their axons toward the striatum. However, the mechanisms by which the dopaminergic axons extend the striatum to innervate their targets remain unclear. We previously showed that paired-cultivation of mesencephalic cells containing dopaminergic neurons with striatal cells leads to the extension of dopaminergic neurites from the mesencephalic cell region to the striatal cell region. The present study shows that dopaminergic neurites extended along striatal neurons in the paired-cultures of mesencephalic cells with striatal cells. The extension of dopaminergic neurites was suppressed by the pharmacological inhibition of integrin α5β1. Using lentiviral vectors, short hairpin RNA (shRNA)-mediated knockdown of integrin α5 in dopaminergic neurons suppressed the neurite outgrowth to the striatal cell region. In contrast, the knockdown of integrin α5 in non-dopaminergic mesencephalic and striatal cells had no effect. Furthermore, overexpression of integrin α5 in dopaminergic neurons differentiated from embryonic stem cells enhanced their neurite outgrowth on striatal cells. These results indicate that integrin α5β1 expression on dopaminergic neurons plays an important role in the dopaminergic neurite outgrowth on striatal neurons. PMID:28176845

  11. 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

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

    PubMed

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

    2016-01-19

    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.

  13. The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening.

    PubMed

    Frimat, Jean-Philippe; Sisnaiske, Julia; Subbiah, Subanatarajan; Menne, Heike; Godoy, Patricio; Lampen, Peter; Leist, Marcel; Franzke, Joachim; Hengstler, Jan G; van Thriel, Christoph; West, Jonathan

    2010-03-21

    We present a rapid, reproducible and sensitive neurotoxicity testing platform that combines the benefits of neurite outgrowth analysis with cell patterning. This approach involves patterning neuronal cells within a hexagonal array to standardize the distance between neighbouring cellular nodes, and thereby standardize the length of the neurite interconnections. This feature coupled with defined assay coordinates provides a streamlined display for rapid and sensitive analysis. We have termed this the network formation assay (NFA). To demonstrate the assay we have used a novel cell patterning technique involving thin film poly(dimethylsiloxane) (PDMS) microcontact printing. Differentiated human SH-SY5Y neuroblastoma cells colonized the array with high efficiency, reliably producing pattern occupancies above 70%. The neuronal array surface supported neurite outgrowth, resulting in the formation of an interconnected neuronal network. Exposure to acrylamide, a neurotoxic reference compound, inhibited network formation. A dose-response curve from the NFA was used to determine a 20% network inhibition (NI(20)) value of 260 microM. This concentration was approximately 10-fold lower than the value produced by a routine cell viability assay, and demonstrates that the NFA can distinguish network formation inhibitory effects from gross cytotoxic effects. Inhibition of the mitogen-activated protein kinase (MAPK) ERK1/2 and phosphoinositide-3-kinase (PI-3K) signaling pathways also produced a dose-dependent reduction in network formation at non-cytotoxic concentrations. To further refine the assay a simulation was developed to manage the impact of pattern occupancy variations on network formation probability. Together these developments and demonstrations highlight the potential of the NFA to meet the demands of high-throughput applications in neurotoxicology and neurodevelopmental biology.

  14. Transthyretin provides trophic support via megalin by promoting neurite outgrowth and neuroprotection in cerebral ischemia

    PubMed Central

    Gomes, J R; Nogueira, RS; Vieira, M; Santos, SD; Ferraz-Nogueira, J P; Relvas, J B; Saraiva, M J

    2016-01-01

    Transthyretin (TTR) is a protein whose function has been associated to binding and distribution of thyroid hormones in the body and brain. However, little is known regarding the downstream signaling pathways triggered by wild-type TTR in the CNS either in neuroprotection of cerebral ischemia or in physiological conditions. In this study, we investigated how TTR affects hippocampal neurons in physiologic/pathologic conditions. Recombinant TTR significantly boosted neurite outgrowth in mice hippocampal neurons, both in number and length, independently of its ligands. This TTR neuritogenic activity is mediated by the megalin receptor and is lost in megalin-deficient neurons. We also found that TTR activates the mitogen-activated protein kinase (MAPK) pathways (ERK1/2) and Akt through Src, leading to the phosphorylation of transcription factor CREB. In addition, TTR promoted a transient rise in intracellular calcium through NMDA receptors, in a Src/megalin-dependent manner. Moreover, under excitotoxic conditions, TTR stimulation rescued cell death and neurite loss in TTR KO hippocampal neurons, which are more sensitive to excitotoxic degeneration than WT neurons, in a megalin-dependent manner. CREB was also activated by TTR under excitotoxic conditions, contributing to changes in the balance between Bcl2 protein family members, toward anti-apoptotic proteins (Bcl2/BclXL versus Bax). Finally, we clarify that TTR KO mice subjected to pMCAO have larger infarcts than WT mice, because of TTR and megalin neuronal downregulation. Our results indicate that TTR might be regarded as a neurotrophic factor, because it stimulates neurite outgrowth under physiological conditions, and promotes neuroprotection in ischemic conditions. PMID:27518433

  15. 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

  16. β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells.

    PubMed

    Salto, Rafael; Vílchez, Jose D; 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.

  17. Prevention of posttraumatic axon sprouting by blocking CRMP2-mediated neurite outgrowth and tubulin polymerization

    PubMed Central

    Wilson, Sarah M.; Xiong, Wenhui; Wang, Yuying; Ping, Xingjie; Head, Jessica D.; Brittain, Joel M.; Gagare, Pravin D.; Ramachandran, P. Veeraraghavan; Jin, Xiaoming; Khanna, Rajesh

    2012-01-01

    Epileptogenesis following traumatic brain injury (TBI) is likely due to a combination of increased excitability, disinhibition, and increased excitatory connectivity via aberrant axon sprouting. Targeting these pathways could be beneficial in the prevention and treatment of posttraumatic epilepsy. Here, we tested this possibility using the novel anticonvulsant (R)-N-benzyl 2-acetamido-3-methoxypropionamide ((R)-lacosamide (LCM) which acts on both voltage-gated sodium channels and collapsin response mediator protein 2 (CRMP2), an axonal growth/guidance protein. LCM inhibited CRMP2-mediated neurite outgrowth, an effect phenocopied by CRMP2 knockdown. Mutation of LCM binding sites in CRMP2 reduced the neurite inhibitory effect of LCM by ~8-fold. LCM also reduced CRMP2-mediated tubulin polymerization. Thus, LCM selectively impairs CRMP2-mediated microtubule polymerization which underlies its neurite outgrowth and branching. To determine whether LCM inhibits axon sprouting in vivo, LCM was injected into rats subjected to partial cortical isolation, an animal model of posttraumatic epileptogenesis that exhibits axon sprouting in cortical pyramidal neurons. Two weeks following injury, excitatory synaptic connectivity of cortical layer V pyramidal neurons was mapped using patch clamp recordings and laser scanning photostimulation of caged glutamate. In comparison to injured control animals, there was a significant decrease in the map size of excitatory synaptic connectivity in LCM-treated rats, suggesting that LCM treatment prevented enhanced excitatory synaptic connectivity due to posttraumatic axon sprouting. These findings suggest, for the first time, that LCM’s mode of action involves interactions with CRMP2 to inhibit posttraumatic axon sprouting. PMID:22433297

  18. Protein Kinase MARK/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity

    PubMed Central

    Biernat, Jacek; Wu, Yong-Zhong; Timm, Thomas; Zheng-Fischhöfer, Qingyi; Mandelkow, Eckhard; Meijer, Laurent; Mandelkow, Eva-Maria

    2002-01-01

    Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. Herein, we report the ability of MARK2 to affect the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites. PMID:12429843

  19. 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

  20. New function of the adaptor protein SH2B1 in brain-derived neurotrophic factor-induced neurite outgrowth.

    PubMed

    Shih, Chien-Hung; Chen, Chien-Jen; Chen, Linyi

    2013-01-01

    Neurite outgrowth is an essential process for the establishment of the nervous system. Brain-derived neurotrophic factor (BDNF) binds to its receptor TrkB and regulates axonal and dendritic morphology of neurons through signal transduction and gene expression. SH2B1 is a signaling adaptor protein that regulates cellular signaling in various physiological processes. The purpose of this study is to investigate the role of SH2B1 in the development of the central nervous system. In this study, we show that knocking down SH2B1 reduces neurite formation of cortical neurons whereas overexpression of SH2B1β promotes the development of hippocampal neurons. We further demonstrate that SH2B1β promotes BDNF-induced neurite outgrowth and signaling using the established PC12 cells stably expressing TrkB, SH2B1β or SH2B1β mutants. Our data indicate that overexpressing SH2B1β enhances BDNF-induced MEK-ERK1/2, and PI3K-AKT signaling pathways. Inhibition of MEK-ERK1/2 and PI3K-AKT pathways by specific inhibitors suggest that these two pathways are required for SH2B1β-promoted BDNF-induced neurite outgrowth. Moreover, SH2B1β enhances BDNF-stimulated phosphorylation of signal transducer and activator of transcription 3 at serine 727. Finally, our data indicate that the SH2 domain and tyrosine phosphorylation of SH2B1β contribute to BDNF-induced signaling pathways and neurite outgrowth. Taken together, these findings demonstrate that SH2B1β promotes BDNF-induced neurite outgrowth through enhancing pathways involved MEK-ERK1/2 and PI3K-AKT.

  1. 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.

  2. 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. Copyright © 2014 Wiley Periodicals, Inc.

  3. 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

  4. 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

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. Regulation of early neurite morphogenesis by the Na+/H+ exchanger NHE1.

    PubMed

    Sin, Wun-Chey; Moniz, David M; Ozog, Mark A; Tyler, Jessica E; Numata, Masayuki; Church, John

    2009-07-15

    The ubiquitously expressed Na(+)/H(+) exchanger NHE1 plays an important role in regulating polarized membrane protrusion and directional motility in non-neuronal cells. Using NGF-differentiated PC12 cells and murine neocortical neurons in vitro, we now show that NHE1 plays a role in regulating early neurite morphogenesis. NHE1 was expressed in growth cones in which it gave rise to an elevated intracellular pH in actively extending neurites. The NHE1 inhibitor cariporide reversibly reduced growth cone filopodia number and the formation and elongation of neurites, especially branches, whereas the transient overexpression of full-length NHE1, but not NHE1 mutants deficient in either ion translocation activity or actin cytoskeletal anchoring, elicited opposite effects. In addition, compared with neocortical neurons obtained from wild-type littermates, neurons isolated from NHE1-null mice exhibited reductions in early neurite outgrowth, an effect that was rescued by overexpression of full-length NHE1 but not NHE1 mutants. Finally, the growth-promoting effects of netrin-1, but not BDNF or IGF-1, were markedly reduced by cariporide in wild-type neocortical neurons and were not observed in NHE1-null neurons. Although netrin-1 failed to increase growth cone intracellular pH or Na(+)/H(+) exchange activity, netrin-1-induced increases in early neurite outgrowth were restored in NHE1-null neurons transfected with full-length NHE1 but not an ion translocation-deficient mutant. Collectively, the results indicate that NHE1 participates in the regulation of early neurite morphogenesis and identify a novel role for NHE1 in the promotion of early neurite outgrowth by netrin-1.

  10. Distinct domains of the limbic system-associated membrane protein (LAMP) mediate discrete effects on neurite outgrowth.

    PubMed

    Eagleson, Kathie L; Pimenta, Aurea F; Burns, Mary M; Fairfull, Liane D; Cornuet, Pamela K; Zhang, Li; Levitt, Pat

    2003-11-01

    The limbic system-associated membrane protein (LAMP) is a glycosylphosphatidylinositol-anchored glycoprotein with three immunoglobulin (Ig) domains that can either enhance or inhibit neurite outgrowth depending upon the neuronal population examined. In the present study, we investigate the domains responsible for these activities. Domain deletion revealed that the N-terminal IgI domain is necessary and sufficient for the neurite-promoting activity observed in hippocampal neurons. In contrast, inhibition of neurite outgrowth in SCG neurons, which is mediated by heterophilic interactions, requires full-length LAMP, although selective inhibition of the second Ig domain, but not the first or third domains, prevented the inhibitory effect. This indicates that the IgII domain of LAMP harbors the neurite-inhibiting activity, but only in the context of the full-length configuration. Covasphere-binding analyses demonstrate IgI/IgI interactions, but no interaction between IgII and any other domain, consistent with the biological activities that each domain mediates. The data suggest that LAMP may serve as a bifunctional guidance molecule, with distinct structural domains contributing to the promotion and inhibition of neurite outgrowth.

  11. 7, 8, 3'-Trihydroxyflavone Promotes Neurite Outgrowth and Protects Against Bupivacaine-Induced Neurotoxicity in Mouse Dorsal Root Ganglion Neurons.

    PubMed

    Shi, Haohong; Luo, Xingjing

    2016-07-02

    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 AND 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.

  12. Enhanced neurite outgrowth of PC-12 cells on graphene-monolayer-coated substrates as biomimetic cues

    NASA Astrophysics Data System (ADS)

    Lee, Jong Ho; Shin, Yong Cheol; Jin, Oh Seong; Han, Dong-Wook; Kang, Seok Hee; Hong, Suck Won; Kim, Jong Man

    2012-11-01

    Neurons are electrically excitable cells that transmit and process information in the nervous system. Recently, the differentiation of human neural stem cells to neurons has been shown to be enhanced on graphene substrates, and differentiated neurons have been shown to be able to still carry electrical signals when stimulated by graphene electrodes. Graphene films grown by using chemical vapor deposition were transferred onto glass coverslips by using the scooping method and were then coated with fetal bovine serum for a neuronal cell culture. The graphene substrates as biomimetic cues have been shown to enhance the neurite outgrowth of PC-12 cells. Our findings suggest that graphene has a unique surface property that can promote neuronal cells, which should open tremendous opportunities in neuroscience, neural engineering and regenerative medicine.

  13. 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. Copyright © 2014 Elsevier B.V. All

  14. Sodium channel activation augments NMDA receptor function and promotes neurite outgrowth in immature cerebrocortical neurons

    PubMed Central

    George, Joju; Dravid, Shashank M.; Prakash, Anand; Xie, Jun; Peterson, Jennifer; Jabba, Sairam V.; Baden, Daniel G.; Murray, Thomas F.

    2009-01-01

    A range of extrinsic signals, including afferent activity, affect neuronal growth and plasticity. Neuronal activity regulates intracellular Ca2+ and activity-dependent calcium signaling has been shown to regulate dendritic growth and branching (Konur and Ghosh, 2005). NMDA receptor (NMDAR) stimulation of Ca2+/calmodulin-dependent protein kinase signaling cascades has moreover been demonstrated to regulate neurite/axonal outgrowth (Wayman et al., 2004). We used a sodium channel activator, brevetoxin (PbTx-2), to explore the relationship between intracellular [Na+] and NMDAR-dependent development. PbTx-2 alone, at a concentration of 30 nM, did not affect Ca2+ dynamics in DIV-2 cerebrocortical neurons; however, this treatment robustly potentiated NMDA-induced Ca2+ influx. The 30 nM PbTx-2 treatment produced a maximum [Na+]i of 16.9 ± 1.5 mM representing an increment of 8.8 ± 1.8 mM over basal. The corresponding membrane potential change produced by 30 nM PbTx-2 was modest and therefore insufficient to relieve the voltage-dependent Mg2+ block of NMDARs. To unambiguously demonstrate the enhancement of NMDA receptor function by PbTx-2, we recorded single-channel currents from cell-attached patches. PbTx-2 treatment was found to increase both the mean open time and open probability of NMDA receptors. These effects of PbTx-2 on NMDA receptor function were dependent on extracellular Na+ and activation of Src kinase. The functional consequences of PbTx-2-induced enhancement of NMDAR function were evaluated in immature cerebrocortical neurons. PbTx-2 concentrations between 3 and 300 nM enhanced neurite outgrowth. Voltage-gated sodium channel activators may accordingly represent a novel pharmacologic strategy to regulate neuronal plasticity through an NMDA receptor and Src family kinase-dependent mechanism. PMID:19279266

  15. Microfluidic Gradients Reveal Enhanced Neurite Outgrowth but Impaired Guidance within 3D Matrices with High Integrin Ligand Densities

    PubMed Central

    Romano, Nicole H.; Lampe, Kyle J.; Xu, Hui; Ferreira, Meghaan M.

    2015-01-01

    The density of integrin-binding ligands in an extracellular matrix (ECM) is known to regulate cell migration speed by imposing a balance of traction forces between the leading and trailing edges of the cell, but the effect of cell-adhesive ligands on neurite chemoattraction is not well understood. We present a platform that combines gradient-generating microfluidic devices with three-dimensional (3D) protein-engineered hydrogels to study the effect of RGD ligand density on neurite pathfinding from chick dorsal root ganglia-derived spheroids. Spheroids are encapsulated in elastin-like polypeptide (ELP) hydrogels presenting either 3.2 or 1.6 mM RGD ligands and exposed to a microfluidic gradient of nerve growth factor (NGF). While the higher ligand density matrix enhanced neurite initiation and persistence of neurite outgrowth, the lower ligand density matrix significantly improved neurite pathfinding and increased the frequency of growth cone turning up the NGF gradient. The apparent trade-off between neurite extension and neurite guidance is reminiscent of the well-known parabolic relationship between cell adhesion and migration speed, implying that a similar matrix-mediated balance of forces regulate neurite elongation and growth cone turning. These results have implications in the design of engineered materials for in vitro models of neural tissue and in vivo nerve guidance channels. PMID:25315156

  16. Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity

    NASA Astrophysics Data System (ADS)

    Leach, Jennie B.; Brown, Xin Q.; Jacot, Jeffrey G.; Di Milla, Paul A.; Wong, Joyce Y.

    2007-06-01

    Rationally designed matrices for nerve tissue engineering and encapsulated cell therapies critically rely on a comprehensive understanding of neural response to biochemical as well as biophysical cues. Whereas biochemical cues are established mediators of neuronal behavior (e.g., outgrowth), physical cues such as substrate stiffness have only recently been recognized to influence cell behavior. In this work, we examine the response of PC12 neurites to substrate stiffness. We quantified and controlled fibronectin density on the substrates and measured multiple neurite behaviors (e.g., growth, branching, neurites per cell, per cent cells expressing neurites) in a large sample population. We found that PC12 neurons display a threshold response to substrate stiffness. On the softest substrates tested (shear modulus ~10 Pa), neurites were relatively few, short in length and unbranched. On stiffer substrates (shear modulus ~102-104 Pa), neurites were longer and more branched and a greater percentage of cells expressed neurites; significant differences in these measures were not found on substrates with a shear modulus >102 Pa. Based on these data and comparisons with published neurobiology and neuroengineering reports of neurite mechanotransduction, we hypothesize that results from studies of neuronal response to compliant substrates are cell-type dependent and sensitive to ligand density, sample size and the range of stiffness investigated.

  17. Heat shock induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway.

    PubMed

    Kano, Yoshio; Nakagiri, Sachiko; Nohno, Tsutomu; Hiragami, Fukumi; Kawamura, Kenji; Kadota, Michiyo; Numata, Keizo; Koike, Yoshihisa; Furuta, Tomohisa

    2004-11-12

    We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.

  18. Electrically conductive biodegradable polymer composite for nerve regeneration: electricity-stimulated neurite outgrowth and axon regeneration.

    PubMed

    Zhang, Ze; Rouabhia, Mahmoud; Wang, Zhaoxu; Roberge, Christophe; Shi, Guixin; Roche, Phillippe; Li, Jiangming; Dao, Lê H

    2007-01-01

    Normal and electrically stimulated PC12 cell cultures and the implantation of nerve guidance channels were performed to evaluate newly developed electrically conductive biodegradable polymer composites. Polypyrrole (PPy) doped by butane sulfonic acid showed a significantly higher number of viable cells compared with PPy doped by polystyrenesulfonate after a 6-day culture. The PC12 cells were left to proliferate for 6 days, and the PPy-coated membranes, showing less initial cell adherence, recorded the same proliferation rate as did the noncoated membranes. Direct current electricity at various intensities was applied to the PC12 cell-cultured conductive membranes. After 7 days, the greatest number of neurites appeared on the membranes with a current intensity approximating 1.7-8.4 microA/cm. Nerve guidance channels made of conductive biodegradable composite were implanted into rats to replace 8 mm of sciatic nerve. The implants were harvested after 2 months and analyzed with immunohistochemistry and transmission electron microscopy. The regenerated nerve tissue displayed myelinated axons and Schwann cells that were similar to those in the native nerve. Electrical stimulation applied through the electrically conductive biodegradable polymers therefore enhanced neurite outgrowth in a current-dependent fashion. The conductive polymers also supported sciatic nerve regeneration in rats.

  19. Potentiation of NGF-induced neurite outgrowth in PC12 cells by papaverine: role played by PLC-γ, IP3 receptors.

    PubMed

    Itoh, Kanako; Ishima, Tamaki; Kehler, Jan; Hashimoto, Kenji

    2011-03-04

    Papaverine, an inhibitor of phosphodiesterase (PDE) 10A, is gaining attention for its potential in the treatment of neuropsychiatric diseases such as schizophrenia. However, the precise mechanisms underlying the putative neuroprotective/neurotrophic actions of papaverine remain unclear. Thus, we investigated the effects of papaverine on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Papaverine potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. In contrast, the selective PDE10A inhibitor MP-10 had no effect on NGF-induced neurite outgrowth. The potentiation of NGF-induced neurite outgrowth by papaverine was blocked by the PLC-γ inhibitor U73122. Furthermore, papaverine's potentiation of NGF-induced neurite outgrowth was also blocked by the co-administration of inositol 1,4,5-trisphosphate (IP(3)) receptor antagonists (xestospongin C and 2-aminoethoxydiphenyl borate (2-APB)) and by reduced expression of IP(3) receptor gene (i.e., itpr1 and itpr3) by siRNA. Our findings suggest that papaverine could potentiate NGF-induced neurite outgrowth, and that activation of PLC-γ and IP(3) receptors might be involved in the mechanism underlying papaverine's potentiation of neurite outgrowth in PC12 cells.

  20. The LIM homeobox gene ceh-14 is required for phasmid function and neurite outgrowth.

    PubMed

    Kagoshima, Hiroshi; Cassata, Giuseppe; Tong, Yong Guang; Pujol, Nathalie; Niklaus, Gisela; Bürglin, Thomas R

    2013-08-15

    Transcription factors play key roles in cell fate specification and cell differentiation. Previously, we showed that the LIM homeodomain factor CEH-14 is expressed in the AFD neurons where it is required for thermotaxis behavior in Caenorhabditis elegans. Here, we show that ceh-14 is expressed in the phasmid sensory neurons, PHA and PHB, a number of neurons in the tail, i.e., PHC, DVC, PVC, PVN, PVQ, PVT, PVW and PVR, as well as the touch neurons. Analysis of the promoter region shows that important regulatory elements for the expression in most neurons reside from -4kb to -1.65kb upstream of the start codon. Further, within the first introns are elements for expression in the hypodermis. Phylogenetic footprinting revealed numerous conserved motifs in these regions. In addition to the existing deletion mutation ceh-14(ch3), we isolated a new allele, ceh-14(ch2), in which only one LIM domain is disrupted. The latter mutant allele is partially defective for thermosensation. Analysis of both mutant alleles showed that they are defective in phasmid dye-filling. However, the cell body, dendritic outgrowth and ciliated endings of PHA and PHB appear normal, indicating that ceh-14 is not required for growth. The loss of a LIM domain in the ceh-14(ch2) allele causes a partial loss-of-function phenotype. Examination of the neurites of ALA and tail neurons using a ceh-14::GFP reporter shows abnormal axonal outgrowth and pathfinding.

  1. 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.

  2. 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

  3. Negletein as a neuroprotectant enhances the action of nerve growth factor and induces neurite outgrowth in PC12 cells.

    PubMed

    Phan, Chia-Wei; Sabaratnam, Vikineswary; Bovicelli, Paolo; Righi, Giuliana; Saso, Luciano

    2016-11-12

    Negletein has been shown to have therapeutic potential for inflammation-associated diseases, but its effect on neurite outgrowth is still unknown. The present study showed that negletein alone did not trigger PC12 cells to differentiate and extend neurites. When compared with the cells in the untreated control, a significant (P < 0.05) induction and a higher neurite outgrowth activity was observed when the cells were cotreated with negletein (10 µM) and a low dose of nerve growth factor (NGF; 5 ng/mL). The neurite outgrowth process was blocked by the tyrosine kinase receptor (Trk) inhibitor, K252a, suggesting that the neuritogenic effect was NGF-dependent. Negletein (10 µM) together with NGF (5 ng/mL) enhanced the phosphorylation of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and cAMP response element-binding protein (CREB). The growth associated protein-43 (GAP-43) and the NGF level were also upregulated by negletein (10 µM) and a low dose of NGF (5 ng/mL). Negletein at nanomolar concentration also was found to be sufficient to mediate the survival of serum-deprived PC12 cells up to 72 h. Taken together, negletein might be useful as an efficient bioactive compound to protect neurons from cell death and promote neuritogenesis. © 2016 BioFactors, 42(6):591-599, 2016.

  4. Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling

    PubMed Central

    Tian, Xiaoxia; Yan, Huijuan; Li, Jiayi; Wu, Shuang; Wang, Junyu; Fan, Lifei

    2017-01-01

    Members of the well-known semaphorin family of proteins can induce both repulsive and attractive signaling in neural network formation and their cytoskeletal effects are mediated in part by small guanosine 5’-triphosphatase (GTPases). The aim of this study was to investigate the cellular role of Rif GTPase in the neurotrophin-induced neurite outgrowth. By using PC12 cells which are known to cease dividing and begin to show neurite outgrowth responding to nerve growth factor (NGF), we found that semaphorin 6A was as effective as nerve growth factor at stimulating neurite outgrowth in PC12 cells, and that its neurotrophic effect was transmitted through signaling by mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K). We further found that neurotrophin-induced neurite formation in PC12 cells could be partially mediated by inhibition of Rif GTPase activity downstream of MAPKs and PI3K signaling. In conclusion, we newly identified Rif as a regulator of the cytoskeletal rearrangement mediated by semaphorins. PMID:28098758

  5. Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling.

    PubMed

    Tian, Xiaoxia; Yan, Huijuan; Li, Jiayi; Wu, Shuang; Wang, Junyu; Fan, Lifei

    2017-01-13

    Members of the well-known semaphorin family of proteins can induce both repulsive and attractive signaling in neural network formation and their cytoskeletal effects are mediated in part by small guanosine 5'-triphosphatase (GTPases). The aim of this study was to investigate the cellular role of Rif GTPase in the neurotrophin-induced neurite outgrowth. By using PC12 cells which are known to cease dividing and begin to show neurite outgrowth responding to nerve growth factor (NGF), we found that semaphorin 6A was as effective as nerve growth factor at stimulating neurite outgrowth in PC12 cells, and that its neurotrophic effect was transmitted through signaling by mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K). We further found that neurotrophin-induced neurite formation in PC12 cells could be partially mediated by inhibition of Rif GTPase activity downstream of MAPKs and PI3K signaling. In conclusion, we newly identified Rif as a regulator of the cytoskeletal rearrangement mediated by semaphorins.

  6. Cdc42hs Facilitates Cytoskeletal Reorganization and Neurite Outgrowth by Localizing the 58-Kd Insulin Receptor Substrate to Filamentous Actin

    PubMed Central

    Govind, Sheila; Kozma, Robert; Monfries, Clinton; Lim, Louis; Ahmed, Sohail

    2001-01-01

    Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein–protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-581267N mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin. PMID:11157984

  7. Nerve growth factor and cytokines mediate lymphoid tissue-induced neurite outgrowth from mouse superior cervical ganglia in vitro.

    PubMed

    Kannan, Y; Bienenstock, J; Ohta, M; Stanisz, A M; Stead, R H

    1996-07-01

    Superior cervical ganglia (SCG) from neonatal mice were cultured with adult murine lymphoid tissue explants in Matrigel (Collaborative Biomedical, Bedford, MA). After 1 and 2 days in culture, many neurites grew toward thymus and spleen. Normal mesenteric lymph node (MLN) induced a smaller effect; however, activated MLN (isolated from mice 10 days after infection with Nippostrongylus brasiliensis; Nb-MLN-10d) caused significantly increased neurite outgrowth. To determine the roles of nerve growth factor (NGF) and cytokines in the promotion of neuritogenesis by lymphoid tissues, anti-NGF and various anti-cytokines were added to cocultures. Anti-NGF inhibited most of the neurite outgrowth toward thymus and spleen but only partially that toward Nb-MLN-10d. Anti-mouse IL-1 beta also significantly reduced the number of neurites growing toward thymus, spleen, and normal MLN. The number of neurites growing toward Nb-MLN-10d was significantly reduced by anti-IL-1 beta, anti-IL-3, anti-IL-6, or anti-GM-CSF. Exogenous IL-1 beta and IL-3 caused neurite outgrowth in single SCG cultures; and the IL-1 beta-, but not the IL-3-, mediated effect was completely blocked by anti-NGF. In one-day thymus/SCG cocultures, endogenous IL-1 was not detectable at concentrations sufficient to cause nerve growth; however, ample NGF was present in the thymic tissues and culture supernatants, but not in SCG. These data suggest that IL-1 mediates NGF production in lymphoid tissues, which in turn induces the growth of sympathetic nerves. Moreover, IL-3, IL-6, or GM-CSF produced during inflammation might also play important roles in the stimulation of nerve growth in vivo.

  8. Induction of Neurite Outgrowth through Contactin and Nr-CAM by Extracellular Regions of Glial Receptor Tyrosine Phosphatase β

    PubMed Central

    Sakurai, Takeshi; Lustig, Marc; Nativ, Moshe; Hemperly, John J.; Schlessinger, Joseph; Peles, Elior; Grumet, Martin

    1997-01-01

    Receptor protein tyrosine phosphatase β (RPTPβ) is expressed as soluble and receptor forms with common extracellular regions consisting of a carbonic anhydrase domain (C), a fibronectin type III repeat (F), and a unique region called S. We showed previously that a recombinant Fc fusion protein with the C domain (βC) binds to contactin and supports neuronal adhesion and neurite growth. As a substrate, βCFS was less effective in supporting cell adhesion, but it was a more effective promoter of neurite outgrowth than βCF. βS had no effect by itself, but it potentiated neurite growth when mixed with βCF. Neurite outgrowth induced by βCFS was inhibited by antibodies against Nr-CAM and contactin, and these cell adhesion molecules formed a complex that bound βCFS. NIH3T3 cells transfected to express βCFS on their surfaces induced neuronal differentiation in culture. These results suggest that binding of glial RPTPβ to the contactin/Nr-CAM complex is important for neurite growth and neuronal differentiation. PMID:9049255

  9. Activation of Aplysia ARF6 induces neurite outgrowth and is sequestered by the overexpression of the PH domain of Aplysia Sec7 proteins.

    PubMed

    Jang, Deok-Jin; Jun, Yong-Woo; Shim, Jaehoon; Sim, Su-Eon; Lee, Jin-A; Lim, Chae-Seok; Kaang, Bong-Kiun

    2017-02-01

    ADP-ribosylation factors (ARFs) are small guanosine triphosphatases of the Ras superfamily involved in membrane trafficking and regulation of the actin cytoskeleton. Aplysia Sec7 protein (ApSec7), a guanine nucleotide exchange factor for ARF1 and ARF6, induces neurite outgrowth and plays a key role in 5-hydroxyltryptamine-induced neurite growth and synaptic facilitation in Aplysia sensory-motor synapses. However, the specific role of ARF6 signaling on neurite outgrowth in Aplysia neurons has not been examined. In the present study, we cloned Aplysia ARF6 (ApARF6) and revealed that an overexpression of enhanced green fluorescent protein (EGFP)-fused constitutively active ApARF6 (ApARF6-Q67L-EGFP) could induce neurite outgrowth in Aplysia sensory neurons. Further, we observed that ApARF6-induced neurite outgrowth was inhibited by the co-expression of a Sec7 activity-deficient mutant of ApSec7 (ApSec7-E159K). The pleckstrin homology domain of ApSec7 may bind to active ApARF6 at the plasma membrane and prevent active ApARF6-induced functions, including intracellular vacuole formation in HEK293T cells. The results of the present study suggest that activation of ARF6 signaling could induce neurite outgrowth in Aplysia neurons and may be involved in downstream signaling of ApSec7-induced neurite outgrowth in Aplysia neurons.

  10. 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...

  11. 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...

  12. Moringa oleifera with promising neuronal survival and neurite outgrowth promoting potentials.

    PubMed

    Hannan, Md Abdul; Kang, Ji-Young; Mohibbullah, Md; Hong, Yong-Ki; Lee, Hyunsook; Choi, Jae-Suk; Choi, In Soon; Moon, Il Soo

    2014-02-27

    Moringa oleifera Lam. (Moringaceae) by virtue of its high nutritional as well as ethnomedical values has been gaining profound interest both in nutrition and medicinal research. The leaf of this plant is used in ayurvedic medicine to treat paralysis, nervous debility and other nerve disorders. In addition, research evidence also suggests the nootropic as well as neuroprotective roles of Moringa oleifera leaf in animal models. The aim of the present study was to evaluate the effect of Moringa oleifera leaf in the primary hippocampal neurons regarding its neurotrophic and neuroprotective properties. The primary culture of embryonic hippocampal neurons was incubated with the ethanol extract of Moringa oleifera leaf (MOE). After an indicated time, cultures were either stained directly with a lipophilic dye, DiO, or fixed and immunolabeled to visualize the neuronal morphology. Morphometric analyses for neurite maturation and synaptogenesis were performed using Image J software. Neuronal viability was evaluated using trypan blue exclusion and lactate dehydrogenase assays. MOE promoted neurite outgrowth in a concentration-dependent manner with an optimal concentration of 30 μg/mL. As a very initial effect, MOE significantly promoted the earlier stages of neuronal differentiation. Subsequently, MOE significantly increased the number and length of dendrites, the length of axon, and the number and length of both dendrite and axonal branches, and eventually facilitated synaptogenesis. The β-carotene, one major compound of MOE, promoted neuritogensis, but the increase was not comparable with the effect of MOE. In addition, MOE supported neuronal survival by protecting neurons from naturally occurring cell death in vitro. Our findings indicate that MOE promotes axodendritic maturation as well as provides neuroprotection suggesting a promising pharmacological importance of this nutritionally and ethnomedically important plant for the well-being of nervous system. Copyright

  13. c-Jun Gene-Modified Schwann Cells: Upregulating Multiple Neurotrophic Factors and Promoting Neurite Outgrowth

    PubMed Central

    Huang, Liangliang; Quan, Xin; Liu, Zhongyang; Ma, Teng; Wu, Yazhen; Ge, Jun; Zhu, Shu; Yang, Yafeng; Liu, Liang; Sun, Zhen

    2015-01-01

    Genetically modified Schwann cells (SCs) that overexpress neurotrophic factors (NFs), especially those that overexpress multiple NFs, hold great potential for promoting nerve regeneration. Currently, only one NF can be upregulated in most genetically modified SCs, and simultaneously upregulating multiple NFs in SCs remains challenging. In this study, we found that the overexpression of c-Jun, a component of the AP-1 transcription factor, effectively upregulated the expression and secretion of multiple NFs, including glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, artemin, leukemia inhibitory factor, and nerve growth factor. The c-Jun gene-modified SCs showed a normal morphology in scanning electron microscopy and fluorescent staining analysis. In addition, the c-Jun-modified SCs showed enhanced proliferation and migration abilities compared with vector control cells. We used transwell chambers to establish coculture systems imitating the in vivo conditions in which transplanted SCs might influence native SCs and neurons. We found that the c-Jun-modified SCs enhanced native SC migration and promoted the proliferation of native SCs in the presence of axons. Further analysis revealed that in the c-Jun group, the average length and the total area of neurites divided by the total area of the explant body were μm 1180±25 and 6.4±0.4, respectively, which were significantly greater compared with the other groups. These findings raise the possibility of constructing an optimal therapeutic alternative for nerve repair using c-Jun-modified SCs, which have the potential to promote axonal regeneration and functional recovery by upregulating multiple NFs. In addition, these cells exhibit enhanced migration and proliferation abilities, enhance the biological functions of native SCs, and promote neurite outgrowth. PMID:25588149

  14. Calcineurin-dependent cofilin activation and increased retrograde actin flow drive 5-HT-dependent neurite outgrowth in Aplysia bag cell neurons.

    PubMed

    Zhang, Xiao-Feng; Hyland, Callen; Van Goor, David; Forscher, Paul

    2012-12-01

    Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca(2+); however, mechanistic roles for Ca(2+) in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca(2+) → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca(2+) release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT-dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.

  15. Neurite outgrowth mediated by the heat shock protein Hsp90α: a novel target for the antipsychotic drug aripiprazole

    PubMed Central

    Ishima, T; Iyo, M; Hashimoto, K

    2012-01-01

    Aripiprazole is an atypical antipsychotic drug approved for the treatment of psychiatric disorders such as schizophrenia, bipolar disorder, major depressive disorder and autism. The drug shows partial agonistic activity at dopamine D2 receptors and 5-hydroxytryptamine (5-HT) 5-HT1A receptors, and antagonistic activity at 5-HT2A receptors. However, the precise mechanistic pathways remain unclear. In this study, we examined the effects of aripiprazole on neurite outgrowth. Aripiprazole significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells, in a concentration-dependent manner. The 5-HT1A receptor antagonist WAY-100635, but not the dopamine D2 receptor antagonist sulpiride, blocked the effects of aripiprazole, although, only partially. Specific inhibitors of inositol 1,4,5-triphosphate (IP3) receptors and BAPTA-AM, a chelator of intracellular Ca2+, blocked the effects of aripiprazole. Moreover, specific inhibitors of several common signaling pathways phospholipase C-γ (PLC-γ), phosphatidylinositol-3 kinase (PI3K), mammalian target of rapamycin, p38 MAPK, c-Jun N-terminal kinase, Akt, Ras, Raf, ERK, MAPK) also blocked the effects of aripiprazole. Using proteomic analysis, we found that aripiprazole significantly increased levels of the heat shock protein Hsp90α in cultured cells. The effects of aripiprazole on NGF-induced neurite outgrowth were significantly attenuated by treatment with Hsp90α RNA interference, but not by the negative control of Hsp90α. These findings suggest that both 5-HT1A receptor activation and Ca2+ signaling via IP3 receptors, as well as their downstream cellular signaling pathways play a role in the promotion of aripiprazole-induced neurite outgrowth. Furthermore, aripiprazole-induced increases in Hsp90α protein expression may form part of the therapeutic mechanism for this drug. PMID:23047241

  16. Antillatoxin, a novel lipopeptide, enhances neurite outgrowth in immature cerebrocortical neurons through activation of voltage-gated sodium channels.

    PubMed

    Jabba, S V; Prakash, A; Dravid, S M; Gerwick, W H; Murray, T F

    2010-03-01

    Antillatoxin (ATX) is a structurally novel lipopeptide that activates voltage-gated sodium channels (VGSC) leading to sodium influx in cerebellar granule neurons and cerebrocortical neurons 8 to 9 days in vitro (Li et al., 2001; Cao et al., 2008). However, the precise recognition site for ATX on the VGSC remains to be defined. Inasmuch as elevation of intracellular sodium ([Na(+)](i)) may increase N-methyl-d-aspartate receptor (NMDAR)-mediated Ca(2+) influx, Na(+) may function as a signaling molecule. We hypothesized that ATX may enhance neurite outgrowth in cerebrocortical neurons by elevating [Na(+)](i) and augmenting NMDAR function. ATX (30-100 nM) robustly stimulated neurite outgrowth, and this enhancement was sensitive to the VGSC antagonist, tetrodotoxin. To unambiguously demonstrate the enhancement of NMDA receptor function by ATX, we recorded single-channel currents from cell-attached patches. ATX was found to increase the open probability of NMDA receptors. Na(+)-dependent up-regulation of NMDAR function has been shown to be regulated by Src family kinase (SFK) (Yu and Salter, 1998). The Src kinase inhibitor PP2 abrogated ATX-enhanced neurite outgrowth, suggesting a SFK involvement in this response. ATX-enhanced neurite outgrowth was also inhibited by the NMDAR antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and the calmodulin-dependent kinase kinase (CaMKK) inhibitor, 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), demonstrating the requirement for NMDAR activation with subsequent downstream engagement of the Ca(2+)-dependent CaMKK pathway. These results with the structurally and mechanistically novel natural product, ATX, confirm and generalize our earlier results with a neurotoxin site 5 ligand. These data suggest that VGSC activators may represent a novel pharmacological strategy to regulate neuronal plasticity through NMDAR-dependent mechanisms.

  17. Pituitary adenylate cyclase-activating polypeptide prevents the effects of ceramides on migration, neurite outgrowth, and cytoskeleton remodeling.

    PubMed

    Falluel-Morel, Anthony; Vaudry, David; Aubert, Nicolas; Galas, Ludovic; Benard, Magalie; Basille, Magali; Fontaine, Marc; Fournier, Alain; Vaudry, Hubert; Gonzalez, Bruno J

    2005-02-15

    During neuronal migration, cells that do not reach their normal destination or fail to establish proper connections are eliminated through an apoptotic process. Recent studies have shown that the proinflammatory cytokine tumor necrosis factor alpha (and its second messengers ceramides) and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) play a pivotal role in the histogenesis of the cerebellar cortex. However, the effects of ceramides and PACAP on migration of cerebellar granule cells have never been investigated. Time-lapse videomicroscopy recording showed that C2-ceramide, a cell-permeable ceramide analog, and PACAP induced opposite effects on cell motility and neurite outgrowth. C2-ceramide markedly stimulated cell movements during the first hours of treatment and inhibited neuritogenesis, whereas PACAP reduced cell migration and promoted neurite outgrowth. These actions of C2-ceramide on cell motility and neurite outgrowth were accompanied by a disorganization of the actin filament network, depolarization of tubulin, and alteration of the microtubule-associated protein Tau. In contrast, PACAP strengthened the polarization of actin at the emergence cone, increased Tau phosphorylation, and abolished C2-ceramide-evoked alterations of the cytoskeletal architecture. The caspase-inhibitor Z-VAD-FMK, like PACAP, suppressed the "dance of the death" provoked by C2-ceramide. Finally, Z-VAD-FMK and the PP2A inhibitor okadaic acid both prevented the impairment of Tau phosphorylation induced by C2-ceramide. Taken together, these data indicate that the reverse actions of C2-ceramide and PACAP on cerebellar granule cell motility and neurite outgrowth are attributable to their opposite effects on actin distribution, tubulin polymerization, and Tau phosphorylation.

  18. 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

  19. The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells

    PubMed Central

    Flowers, Brittany M.; Rusnak, Lauren E.; Wong, Kristen E.; Banks, Dallas A.; Munyikwa, Michelle R.; McFarland, Alexander G.; Hinton, Shantá D.

    2014-01-01

    The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and

  20. 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

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

    PubMed

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

    2012-07-19

    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. 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. 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. P. giganteus contains bioactive compounds that mimic NGF and are responsible for neurite stimulation. Hence, this mushroom may be

  2. MARK2 Rescues Nogo-66-Induced Inhibition of Neurite Outgrowth via Regulating Microtubule-Associated Proteins in Neurons In Vitro.

    PubMed

    Zuo, Yu-Chao; Xiong, Nan-Xiang; Shen, Jian-Ying; Yu, Hua; Huang, Yi-Zhi; Zhao, Hong-Yang

    2016-11-01

    The ability of neurons in the adult mammalian central nervous system (CNS) to regenerate after injury is limited by inhibitors in CNS myelin. Nogo-66 is the most important myelin inhibitor but the mechanisms of Nogo-66 inhibition of neurite outgrowth remain poorly understood. Particularly, the relationship between Nogo-66 and microtubule-affinity regulating kinase 2 (MARK2) has not been examined. This study investigated the role of MARK2 in Nogo-66 inhibition and the function of MARK2 in neurite elongation in neurons in vitro. MARK2 and phosphorylated MARK2 at Ser212 (p-Ser212) alterations in Neuro 2a cells were assessed at different Nogo-66 exposure times; the relationships between MARK2 and microtubule-associated proteins (MAPs) were determined via the overexpression or interference of MARK2. Our study reports that Nogo-66 inhibited the expression of total MARK2 but also reduced Ser212 phosphorylation of MARK2, whereas levels of MAP1-b and tau varied depending on MARK2 overexpression or reduced expression. Furthermore, MARK2 increased the proportion of tyrosinated α-tubulin, thereby disrupting the stability of tubulin, most likely affecting axonal growth. In line with these results, overexpression of MARK2 promoted neurite elongation and therefore is able to rescue the inhibitory effect of Nogo-66 on neurite growth. In conclusion, the intracellular PKB/MARK2/MAPs/α-tubulin pathway appears to be essential for neurite elongation in neurons in vitro. These results suggest a critical role for MARK2 in overcoming Nogo-66-induced inhibition of axon outgrowth in neurons. Pharmacological activators of MARK2 may be applicable to promote successful axonal outgrowth following many types of CNS injuries.

  3. A tenascin-C mimetic peptide amphiphile nanofiber gel promotes neurite outgrowth and cell migration of neurosphere-derived cells.

    PubMed

    Berns, Eric J; Álvarez, Zaida; Goldberger, Joshua E; Boekhoven, Job; Kessler, John A; Kuhn, H Georg; Stupp, Samuel I

    2016-06-01

    Biomimetic materials that display natural bioactive signals derived from extracellular matrix molecules like laminin and fibronectin hold promise for promoting regeneration of the nervous system. In this work, we investigated a biomimetic peptide amphiphile (PA) presenting a peptide derived from the extracellular glycoprotein tenascin-C, known to promote neurite outgrowth through interaction with β1 integrin. The tenascin-C mimetic PA (TN-C PA) was found to self-assemble into supramolecular nanofibers and was incorporated through co-assembly into PA gels formed by highly aligned nanofibers. TN-C PA content in these gels increased the length and number of neurites produced from neurons differentiated from encapsulated P19 cells. Furthermore, gels containing TN-C PA were found to increase migration of cells out of neurospheres cultured on gel coatings. These bioactive gels could serve as artificial matrix therapies in regions of neuronal loss to guide neural stem cells and promote through biochemical cues neurite extension after differentiation. One example of an important target would be their use as biomaterial therapies in spinal cord injury. Tenascin-C is an important extracellular matrix molecule in the nervous system and has been shown to play a role in regenerating the spinal cord after injury and guiding neural progenitor cells during brain development, however, minimal research has been reported exploring the use of biomimetic biomaterials of tenascin-C. In this work, we describe a selfassembling biomaterial system in which peptide amphiphiles present a peptide derived from tenascin-C that promotes neurite outgrowth. Encapsulation of neurons in hydrogels of aligned nanofibers formed by tenascin-C-mimetic peptide amphiphiles resulted in enhanced neurite outgrowth. Additionally, these peptide amphiphiles promoted migration of neural progenitor cells cultured on nanofiber coatings. Tenascin-C biomimetic biomaterials such as the one described here have

  4. Neurite Outgrowth in PC12 Cells Stimulated by Components from Dendranthema × grandiflorum cv. “Mottenohoka” Is Enhanced by Suppressing Phosphorylation of p38MAPK

    PubMed Central

    Kimura, Hirokazu; Tsukagoshi, Hiroyuki; Kozawa, Kunihisa; Koketsu, Mamoru; Ninomiya, Masayuki; Furukawa, Shoei

    2013-01-01

    Components from Dendranthema × grandiflorum cv. “Mottenohoka” that promote neurite outgrowth of PC12 cells were identified and the mechanism of neurite outgrowth stimulated by isolated components was studied. Components that promoted the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) of PC12 cells were isolated. From various structural analyses, the active components were identified as acacetin and luteolin. The effects of acacetin or luteolin on PC12 cells were evaluated by electro-blotting and immunostaining. Slight neurite outgrowth in PC12 cells was observed within 2 days of culture after stimulation by luteolin or acacetin. However, NGF-stimulation induced remarkable neurite outgrowth in comparison. Neurite outgrowth by luteolin or acacetin was significantly enhanced by pretreatment with SB203580 (a p38MAPK inhibitor). The results of this study into the phosphorylation of ERK 1/2 and p38MAPK by flavonoids suggest that the inhibition of p38MAPK phosphorylation may effectively enhance neurite outgrowth. PMID:23554829

  5. Effect of HDAC inhibitors on neuroprotection and neurite outgrowth in primary rat cortical neurons following ischemic insult.

    PubMed

    Hasan, Mohammad Rakibul; Kim, Ji-Hye; Kim, Youn Jung; Kwon, Kyoung Ja; Shin, Chan Young; Kim, Hahn Young; Han, Seol-Heui; Choi, Dong-Hee; Lee, Jongmin

    2013-09-01

    Histone deacetylase inhibitors (HDACi)-valproic acid (VPA) and trichostatin A (TSA) promote neurogenesis, neurite outgrowth, synaptic plasticity and neuroprotection. In this study, we investigated whether VPA and TSA promote post-ischemic neuroprotection and neuronal restoration in rat primary cortical neurons. On 6 days in vitro (DIV), cortical neurons were exposed to oxygen-glucose deprivation for 90 min. Cells were returned to normoxic conditions and cultured for 1, 3, or 7 days with or without VPA and TSA. Control cells were cultured in normoxic conditions only. On 7, 9, and 13 DIV, cells were measured neurite outgrowth using the Axiovision program and stained with Tunel staining kit. Microtubule associated protein-2 immunostaining and tunel staining showed significant recovery of neurite outgrowth and post-ischemic neuronal death by VPA or TSA treatment. We also determined levels of acetylated histone H3, PSD95, GAP 43 and synaptophysin. Significant increases in all three synaptic markers and acetylated histone H3 were observed relative to non-treated cells. Post-ischemic HDACi treatment also significantly raised levels of brain derived neurotrophic factor (BDNF) expression and secreted BDNF. Enhanced BDNF expression by HDACi treatment might have been involved in the post-ischemic neuroprotection and neuronal restorative effects. Our findings suggest that both VPA and TSA treatment during reoxygenation after ischemia may help post-ischemic neuroprotection and neuronal regeneration via increased BDNF expression and activation.

  6. A Sonic hedgehog coreceptor, BOC regulates neuronal differentiation and neurite outgrowth via interaction with ABL and JNK activation.

    PubMed

    Vuong, Tuan Anh; Leem, Young-Eun; Kim, Bok-Geon; Cho, Hana; Lee, Sang-Jin; Bae, Gyu-Un; Kang, Jong-Sun

    2017-01-01

    Neurite outgrowth is a critical step for neurogenesis and remodeling synaptic circuitry during neuronal development and regeneration. An immunoglobulin superfamily member, BOC functions as Sonic hedgehog (Shh) coreceptor in canonical and noncanonical Shh signaling in neuronal development and axon outgrowth/guidance. However signaling mechanisms responsible for BOC action during these processes remain unknown. In our previous studies, a multiprotein complex containing BOC and a closely related protein CDO promotes myogenic differentiation through activation of multiple signaling pathways, including non-receptor tyrosine kinase ABL. Given that ABL and Jun. N-terminal kinase (JNK) are implicated in actin cytoskeletal dynamics required for neurogenesis, we investigated the relationship between BOC, ABL and JNK during neuronal differentiation. Here, we demonstrate that BOC and ABL are induced in P19 embryonal carcinoma (EC) cells and cortical neural progenitor cells (NPCs) during neuronal differentiation. BOC-depleted EC cells or Boc(-/-) NPCs exhibit impaired neuronal differentiation with shorter neurite formation. BOC interacts with ABL through its putative SH2 binding domain and seems to be phosphorylated in an ABL activity-dependent manner. Unlike wildtype BOC, ABL-binding defective BOC mutants exhibit impaired JNK activation and neuronal differentiation. Finally, Shh treatment enhances JNK activation which is diminished by BOC depletion. These data suggest that BOC interacts with ABL and activates JNK thereby promoting neuronal differentiation and neurite outgrowth. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  8. Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms.

    PubMed

    Wang, Ze-Jian; Nie, Bao-Ming; Chen, Hong-Zhuan; Lu, Yang

    2006-01-05

    Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.

  9. Semiconductor Nanomembrane Tubes: Three-Dimensional Confinement for Controlled Neurite Outgrowth

    PubMed Central

    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.

    2013-01-01

    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

  10. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Sominone enhances neurite outgrowth and spatial memory mediated by the neurotrophic factor receptor, RET.

    PubMed

    Tohda, Chihiro; Joyashiki, Eri

    2009-08-01

    Orally administered withanoside IV (a compound isolated from the roots of Withania somnifera) improved memory deficits in mice with a model of Alzheimer's disease induced by the amyloid peptide Abeta(25-35). Sominone, an aglycone of withanoside IV, was identified as an active metabolite after oral administration of withanoside IV. We aimed to identify receptors or associated molecules of sominone, and to investigate the effects of sominone on memory in normal mice. Phosphorylation levels of 71 molecules were compared between control and sominone-stimulated cortical cultured cells to search for target molecules of sominone. Object location memory and neurite density in the brain were evaluated in sominone-injected mice. Phosphorylation of RET (a receptor for the glial cell line-derived neurotrophic factor, GDNF) was increased in neurons by sominone, without affecting the synthesis and secretion of GDNF. Knockdown of RET prevented sominone-induced outgrowths of axons and dendrites. After a single i.p. injection of sominone into normal mice, they could better memorize scenery information than control mice. Sixty minutes after sominone injection, RET phosphorylation was increased, particularly in the hippocampus of mice. After the memory tests, the densities of axons and dendrites were increased in the hippocampus by sominone administration. Sominone could reinforce the morphological plasticity of neurons by activation of the RET pathway and thus enhance memory. Sominone, a compound with low molecular weight, may be a GDNF-independent stimulator of the RET pathway and/or a novel modulator of RET signalling.

  12. Cobalt chloride induces neurite outgrowth in rat pheochromocytoma PC-12 cells through regulation of endothelin-2/vasoactive intestinal contractor.

    PubMed

    Kotake-Nara, Eiichi; Takizawa, Satoshi; Quan, Jiexia; Wang, Hongyu; Saida, Kaname

    2005-08-15

    We investigated whether endothelin-2/vasoactive intestinal contractor (ET-2/VIC) gene expression, upregulated by hypoxia in cancer cells, was associated with differentiation in neuronal cells. RT-PCR analysis, morphological observations, and immunostaining revealed that CoCl2, a hypoxic mimetic agent, at 200 microM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced neurite outgrowth in PC-12 rat pheochromocytoma cells. These effects induced by 200 microM CoCl2 were completely inhibited by the antioxidant N-acetyl cysteine at 20 mM. In addition, CoCl2 increased the level of intracellular reactive oxygen species (ROS) at an early stage. Furthermore, interleukin (IL)-6 gene expression was upregulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by ROS may be associated with neuronal differentiation through the regulation of IL-6. When the cells were treated with 500 microM CoCl2 for 24 hr, however, ET-2/VIC gene expression disappeared, IL-6 gene expression was downregulated, and necrosis was subsequently induced in the PC-12 cells.

  13. Gene expression analysis of laminin-1-induced neurite outgrowth in human mesenchymal stem cells derived from bone marrow.

    PubMed

    Mruthyunjaya, S; Parveen, D; Shah, Reecha D; Manchanda, Rumma; Godbole, Ravibhushan; Vasudevan, Madavan; Shastry, Padma

    2015-02-01

    The mechanisms underlying the differentiation of Mesenchymal stem cells (MSCs) toward neuronal cell type are not clearly understood. Earlier, we reported that laminin-1 induces neurite outgrowth in human MSCs via c-Jun/AP-1 activation through ERK, JNK, and Akt pathways. In this study, we demonstrate that laminin-1 increases the expression of proneural gene, neuroD1 and induces the expression of immediate-early biomarkers of neuronal cell-programming-Egr1, Egr3, PC3, and PC4. Gene expression profiling of MSCs cultured on laminin-1 and Poly-l-lysine for 12 h revealed differential regulation of 267 genes (>1.5 fold, p < 0.05), predominantly in the category of nervous system development and affected the pathways involved in TGF-β/TNF-α signaling, regulation of MAPK and JNK cascade. Data for 11 selected genes related to nervous system development was validated by real time PCR. Transcriptional regulatory network analysis revealed c-Jun as the key transcription factor regulating majority of differentially expressed genes and identified Disrupted in schizophrenia 1, as a novel target of c-Jun. Modeling and analysis of biological network showed selective induction of Growth Arrest and DNA damage 45 (GADD45B) and repression of NF-κB inhibitor A (NFκBIA). Collectively, our findings provide the basis for understanding the molecular mechanisms associated with laminin-1-induced neurogenic expression in MSCs. © 2014 Wiley Periodicals, Inc.

  14. A study of the effects of flux density and frequency of pulsed electromagnetic field on neurite outgrowth in PC12 cells.

    PubMed

    Zhang, Yang; Ding, Jun; Duan, Wei

    2006-01-01

    The aim of this study was to investigate the influence of pulsed electromagnetic fields with various flux densities and frequencies on neurite outgrowth in PC12 rat pheochromocytoma cells. We have studied the percentage of neurite-bearing cells, average length of neurites and directivity of neurite outgrowth in PC12 cells cultured for 96 hours in the presence of nerve growth factor (NGF). PC12 cells were exposed to 50 Hz pulsed electromagnetic fields with a flux density of 1.37 mT, 0.19 mT and 0.016 mT respectively. The field was generated through a Helmholtz coil pair housed in one incubator and the control samples were placed in another identical incubator. It was found that exposure to both a relatively high flux density (1.37 mT) and a medium flux density (0.19 mT) inhibited the percentage of neurite-bearing cells and promoted neurite length significantly. Exposure to high flux density (1.37 mT) also resulted in nearly 20% enhancement of neurite directivity along the field direction. However, exposure to low flux density field (0.016 mT) had no detectable effect on neurite outgrowth. We also studied the effect of frequency at the constant flux density of 1.37 mT. In the range from 1 approximately 100 Hz, only 50 and 70 Hz pulse frequencies had significant effects on neurite outgrowth. Our study has shown that neurite outgrowth in PC12 cells is sensitive to flux density and frequency of pulsed electromagnetic field.

  15. Gangliosides and Nogo receptors independently mediate myelin-associated glycoprotein inhibition of neurite outgrowth in different nerve cells.

    PubMed

    Mehta, Niraj R; Lopez, Pablo H H; Vyas, Alka A; Schnaar, Ronald L

    2007-09-21

    In the injured nervous system, myelin-associated glycoprotein (MAG) on residual myelin binds to receptors on axons, inhibits axon outgrowth, and limits functional recovery. Conflicting reports identify gangliosides (GD1a and GT1b) and glycosylphosphatidylinositol-anchored Nogo receptors (NgRs) as exclusive axonal receptors for MAG. We used enzymes and pharmacological agents to distinguish the relative roles of gangliosides and NgRs in MAG-mediated inhibition of neurite outgrowth from three nerve cell types, dorsal root ganglion neurons (DRGNs), cerebellar granule neurons (CGNs), and hippocampal neurons. Primary rat neurons were cultured on control substrata and substrata adsorbed with full-length native MAG extracted from purified myelin. The receptors responsible for MAG inhibition of neurite outgrowth varied with nerve cell type. In DRGNs, most of the MAG inhibition was via NgRs, evidenced by reversal of inhibition by phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves glycosylphosphatidylinositol anchors, or by NEP1-40, a peptide inhibitor of NgR. A smaller percentage of MAG inhibition of DRGN outgrowth was via gangliosides, evidenced by partial reversal by addition of sialidase to cleave GD1a and GT1b or by P4, an inhibitor of ganglioside biosynthesis. Combining either PI-PLC and sialidase or NEP1-40 and P4 was additive. In contrast to DRGNs, in CGNs MAG inhibition was exclusively via gangliosides, whereas inhibition of hippocampal neuron outgrowth was mostly reversed by sialidase or P4 and only modestly reversed by PI-PLC or NEP1-40 in a non-additive fashion. A soluble proteolytic fragment of native MAG, dMAG, also inhibited neurite outgrowth. In DRGNs, dMAG inhibition was exclusively NgR-dependent, whereas in CGNs it was exclusively ganglioside-dependent. An inhibitor of Rho kinase reversed MAG-mediated inhibition in all nerve cells, whereas a peptide inhibitor of the transducer p75(NTR) had cell-specific effects quantitatively similar to Ng

  16. The death receptor antagonist FAIM promotes neurite outgrowth by a mechanism that depends on ERK and NF-κB signaling

    PubMed Central

    Sole, Carme; Dolcet, Xavier; Segura, Miguel F.; Gutierrez, Humberto; Diaz-Meco, Maria-Teresa; Gozzelino, Raffaella; Sanchis, Daniel; Bayascas, Jose R.; Gallego, Carme; Moscat, Jorge; Davies, Alun M.; Comella, Joan X.

    2004-01-01

    Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. We show that FAIM overexpression fails to rescue neurons from trophic factor deprivation, but exerts a marked neurite growth–promoting action in different neuronal systems. Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells. FAIM overexpression promoted NF-κB activation, and blocking this activation by using a super-repressor IκBα or by carrying out experiments using cortical neurons from mice that lack the p65 NF-κB subunit prevented FAIM-induced neurite outgrowth. The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras–ERK pathway. Finally, we show that FAIM interacts with both Trk and p75 neurotrophin receptor NGF receptors in a ligand-dependent manner. These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras–ERK pathway and NF-κB. PMID:15520226

  17. Soluble cpg15 from Astrocytes Ameliorates Neurite Outgrowth Recovery of Hippocampal Neurons after Mouse Cerebral Ischemia.

    PubMed

    Zhao, Jing-Jing; Hu, Jie-Xian; Lu, De-Xin; Ji, Chun-Xia; Qi, Yao; Liu, Xiao-Yan; Sun, Feng-Yan; Huang, Fang; Xu, Ping; Chen, Xian-Hua

    2017-02-08

    The present study focuses on the function of cpg15, a neurotrophic factor, in ischemic neuronal recovery using transient global cerebral ischemic (TGI) mouse model and oxygen-glucose deprivation (OGD)-treated primary cultured cells. The results showed that expression of cpg15 proteins in astrocytes, predominantly the soluble form, was significantly increased in mouse hippocampus after TGI and in the cultured astrocytes after OGD. Addition of the medium from the cpg15-overexpressed astrocytic culture into the OGD-treated hippocampal neuronal cultures reduces the neuronal injury, whereas the recovery of neurite outgrowths of OGD-injured neurons was prevented when cpg15 in the OGD-treated astrocytes was knocked down, or the OGD-treated-astrocytic medium was immunoadsorbed by cpg15 antibody. Furthermore, lentivirus-delivered knockdown of cpg15 expression in mouse hippocampal astrocytes diminishes the dendritic branches and exacerbates injury of neurons in CA1 region after TGI. In addition, treatment with inhibitors of MEK1/2, PI3K, and TrkA decreases, whereas overexpression of p-CREB, but not dp-CREB, increases the expression of cpg15 in U118 or primary cultured astrocytes. Also, it is observed that the Flag-tagged soluble cpg15 from the astrocytes transfected with Flag-tagged cpg15-expressing plasmids adheres to the surface of neuronal bodies and the neurites. In conclusion, our results suggest that the soluble cpg15 from astrocytes induced by ischemia could ameliorate the recovery of the ischemic-injured hippocampal neurons via adhering to the surface of neurons. The upregulated expression of cpg15 in astrocytes may be activated via MAPK and PI3K signal pathways, and regulation of CREB phosphorylation.SIGNIFICANCE STATEMENT Neuronal plasticity plays a crucial role in the amelioration of neurological recovery of ischemic injured brain, which remains a challenge for clinic treatment of cerebral ischemia. cpg15 as a synaptic plasticity-related factor may participate in

  18. Oligodendrocyte precursor cells differentially expressing Nogo-A but not MAG are more permissive to neurite outgrowth than mature oligodendrocytes.

    PubMed

    Ma, Zhengwen; Cao, Qilin; Zhang, Liqun; Hu, Jianguo; Howard, Russell M; Lu, Peihua; Whittemore, Scott R; Xu, Xiao-Ming

    2009-05-01

    Grafting oligodendrocyte precursor cells (OPCs) has been used as a strategy to repair demyelination of the central nervous system (CNS). Whether OPCs can promote CNS axonal regeneration remains to be tested. If so, they should be permissive to axonal growth and may express less inhibitory molecules on their surface. Here we examined the expression of two oligodendrocyte-associated myelin inhibitors Nogo-A and myelin-associated glycoprotein (MAG) during oligodendrogliogenesis and tested their abilities to promote neurite outgrowth in vitro. Whereas the intracellular domain of Nogo-A was consistently expressed throughout oligodendrocyte differentiation, MAG was expressed only at later stages. Furthermore, the membrane-associated extracellular domain of Nogo-A was not expressed in OPCs but expressed in mature oligodendrocytes. In a dorsal root ganglion (DRG) and OPC/oligodendrocyte co-culture model, significantly greater DRG neurite outgrowth onto OPC monolayer than mature oligodendrocyte was found (1042+/-123 vs. 717+/-342 micrometer; p=0.011). Moreover, DRG neurites elongated as fasciculated fiber tracts and contacted directly on OPCs (133+/-37 cells/fascicle). In contrast, few, if any, direct contacts were found between DRG neurites and mature oligodendrocytes (5+/-3 cells/fascicle, p<0.001). In fact, acellular spaces were found between neurites and surrounding mature oligodendrocytes in contrast to the lack of such spaces in OPC/DRG coculture (51.1+/-16.5 vs. 2.4+/-3.9 micrometer; p<0.001). Thus, OPCs expressing neither extracellular domain of Nogo-A nor MAG are significantly more permissive than mature oligodendrocytes expressing both. Grafting OPCs may thus represent a feasible strategy to foster CNS axonal regeneration.

  19. Runx1 contributes to the functional switching of bone morphogenetic protein 4 (BMP4) from neurite outgrowth promoting to suppressing in dorsal root ganglion.

    PubMed

    Yoshikawa, Masaaki; Masuda, Tomoyuki; Kobayashi, Azusa; Senzaki, Kouji; Ozaki, Shigeru; Aizawa, Shin; Shiga, Takashi

    2016-04-01

    The runt-related transcription factor Runx1 regulates cell-type specification and axonal projections of nociceptive dorsal root ganglion (DRG) neurons, whereas bone morphogenetic protein 4 (BMP4) is required for axonal growth during neuronal development. Although Runx1 has been shown to be involved in BMP4 signaling in non-neural tissues, the Runx1 function in BMP4-dependent regulation of neuronal development is unclear. To investigate interactions between Runx1 and BMP4 in neurite outgrowth, we cultured DRGs from wild-type and Runx1-deficient mouse embryos in the presence or absence of BMP4. Neurite outgrowth was decreased in BMP4-treated wild-type DRGs and untreated Runx1-deficient DRGs, suggesting the inhibitory effect of BMP4 and facilitatory effect of Runx1 on neurite outgrowth. In addition, the combination of BMP4 treatment and Runx1 deficiency increased neurite outgrowth, suggesting that Runx1 is required for BMP4-induced suppression of neurite outgrowth and that the loss of Runx1 results in a functional switch of BMP4 from neurite growth suppressing to neurite growth promoting. Both BMP4 treatment and Runx1 deficiency increased calcitonin gene-related peptide (CGRP)-positive neurons, and CGRP expression was not increased by BMP4 treatment in Runx1-deficient mice, suggesting that Runx1 contributes to BMP4-induced CGRP expression in DRG neurons. Thus, Runx1 contributes to BMP4 regulation of neurite outgrowth and CGRP expression in DRG and may control BMP4 functional switching during embryogenesis.

  20. Flavonoids isolated from Rumex aquaticus exhibit neuroprotective and neurorestorative properties by enhancing neurite outgrowth and synaptophysin.

    PubMed

    Orbán-Gyapai, Orsolya; Raghavan, Aparna; Vasas, Andrea; Forgo, Peter; Hohmann, Judit; Shah, Zahoor A

    2014-01-01

    There is heightened interest in the field of stroke recovery as there is need for agents that would prevent the debilitating effects of the disorder, thereby tremendously reducing the societal and economic costs associated with it. In this study, the isolation of two flavonoids--quercetin-3-O-galactoside (1) and quercetin-3-O-arabinoside (2)--from Rumex aquaticus (western dock) and their neuroprotective effects were reported in the oxygen-glucose deprivation (OGD) model of in vitro ischemia using rat pheochromocytoma (PC12) cell line. Bioassay-guided fractionation of the ethyl-acetate extract of Rumex aquaticus L. afforded the isolation of compounds 1 and 2. The structures of compounds were established on the basis of spectroscopic analyses (UV, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). Both compounds were isolated for the first time from this species. In the course of the pharmacological experiments it was detected that these flavonoids at 10 µM concentration significantly improved cell survival in the oxygen-glucose deprivation model of ischemia. Moreover, they also increased neurite outgrowth in differentiated PC12 cells subjected to ischemic insult. Investigations on the cellular mechanism for the observed effect revealed that compound 1 (10 µM) enhances the expression of synaptophysin - a marker of synapses, and an indicator of synaptic plasticity. Rapid restoration of neurological function following injury is paramount to the prevention of debilitating consequences of ischemic stroke. This combination of neuroprotection and neuritogenic potential could be particularly useful in the recovery phase of stroke.

  1. The BMP coreceptor RGMb promotes while the endogenous BMP antagonist noggin reduces neurite outgrowth and peripheral nerve regeneration by modulating BMP signaling.

    PubMed

    Ma, Chi H E; Brenner, Gary J; Omura, Takao; Samad, Omar A; Costigan, Michael; Inquimbert, Perrine; Niederkofler, Vera; Salie, Rishard; Sun, Chia Chi; Lin, Herbert Y; Arber, Silvia; Coppola, Giovanni; Woolf, Clifford J; Samad, Tarek A

    2011-12-14

    Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb(-/-) mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb(-/-) mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin.

  2. Neurites outgrowth and amino acids levels in goldfish retina under hypo-osmotic or hyper-osmotic conditions.

    PubMed

    Cubillán, Lisbeth; Obregón, Francisco; Lima, Lucimey

    2012-02-01

    Amino acids are known to play relevant roles as osmolytes in various tissues, including the retina. Taurine is one of these active molecules. In addition, taurine stimulates outgrowth from the goldfish retina by mechanisms that include extracellular matrix, calcium fluxes and protein phosphorylation. The present report aims to explore the effect of medium osmolarity on goldfish retinal outgrowth and the possible modifications produced by changing eye osmolarity on amino acid levels in the retina. Goldfish retinal explants were obtained 10 days after crush of the optic nerve and cultured under iso-, hypo- or hyper-osmotic conditions. Hypo-osmotic medium was prepared by diluting the solutions 10% twice, preserving fetal calf serum concentration. Hyper-osmotic medium was done by adding 50 or 100 mM urea or mannitol. Evaluation of length and density of neurites was performed 5 days after plating. Outgrowth was reduced in hypo- and in hyper-osmotic conditions. Taurine, 4 mM, increased length and density of neurites in iso-osmotic, and produced stimulatory effects under both hyper-osmotic conditions. The in vivo modification of osmolarity by intraocular injection of water or 100 mM urea modified levels of free amino acids in the retina. Taurine and aspartate retinal levels increased in a time-dependent manner after hypo- and hyper-osmotic solution injections. Serine, threonine, arginine, γ-aminobutyric acid, alanine and tyrosine were elevated in hyper-osmotic conditions. Outgrowth in vitro, after in vivo osmolarity changes, was higher in the absence of taurine, but did not increase in the presence of the amino acid. The fact that certain outgrowth took place in these conditions support that the impairment was not due to tissue damage. Rather, the effects might be related to the cascade of kinase events described during osmolarity variations. The time course under these conditions produced adjustments in ganglion cells probably related to taurine transporter, and

  3. PAd-shRNA-PTN reduces pleiotrophin of pancreatic cancer cells and inhibits neurite outgrowth of DRG

    PubMed Central

    Yao, Jun; Zhang, Min; Ma, Qing-Yong; Wang, Zheng; Wang, Lian-Cai; Zhang, Dong

    2011-01-01

    AIM: To investigate the silencing effects of pAd-shRNA-pleiotrophin (PTN) on PTN in pancreatic cancer cells, and to observe the inhibition of pAd-shRNA-PTN on neurite outgrowth from dorsal root ganglion (DRG) neurons in vitro. METHODS: PAd-shRNA-PTN was used to infect pancreatic cancer BxPC-3 cells; assays were conducted for knockdown of the PTN gene on the 0th, 1st, 3rd, 5th, 7th and 9th d after infection using immunocytochemistry, real-time quantitative polymerase chain reaction (PCR), and Western blotting analysis. The morphologic changes of cultured DRG neurons were observed by mono-culture of DRG neurons and co-culture with BXPC-3 cells in vitro. RESULTS: The real-time quantitative PCR showed that the inhibition rates of PTN mRNA expression in the BxPC-3 cells were 20%, 80%, 50% and 25% on the 1st, 3rd, 5th and 7th d after infection. Immunocytochemistry and Western blotting analysis also revealed the same tendency. In contrast to the control, the DRG neurons co-cultured with the infected BxPC-3 cells shrunk; the number and length of neurites were significantly decreased. CONCLUSION: Efficient and specific knockdown of PTN in pancreatic cancer cells and the reduction in PTN expression resulted in the inhibition of neurite outgrowth from DRG neurons. PMID:21677838

  4. Controlled release of 6-aminonicotinamide from aligned, electrospun fibers alters astrocyte metabolism and dorsal root ganglia neurite outgrowth

    NASA Astrophysics Data System (ADS)

    Schaub, Nicholas J.; Gilbert, Ryan J.

    2011-08-01

    Following central nervous system (CNS) injury, activated astrocytes form a glial scar that inhibits the migration of axons ultimately leading to regeneration failure. Biomaterials developed for CNS repair can provide local delivery of therapeutics and/or guidance mechanisms to encourage cell migration into damaged regions of the brain or spinal cord. Electrospun fibers are a promising type of biomaterial for CNS injury since these fibers can direct cellular and axonal migration while slowly delivering therapy to the injury site. In this study, it was hypothesized that inclusion of an anti-metabolite, 6-aminonicotinamide (6AN), within poly-l-lactic acid electrospun fibers could attenuate astrocyte metabolic activity while still directing axonal outgrowth. Electrospinning parameters were varied to produce highly aligned electrospun fibers that contained 10% or 20% (w/w) 6AN. 6AN release from the fiber substrates occurred continuously over 2 weeks. Astrocytes placed onto drug-releasing fibers were less active than those cultured on scaffolds without 6AN. Dorsal root ganglia placed onto control and drug-releasing scaffolds were able to direct neurites along the aligned fibers. However, neurite outgrowth was stunted by fibers that contained 20% 6AN. These results show that 6AN release from aligned, electrospun fibers can decrease astrocyte activity while still directing axonal outgrowth.

  5. A novel role for PTEN in the inhibition of neurite outgrowth by Myelin-associated glycoprotein in cortical neurons

    PubMed Central

    Perdigoto, Ana Luisa; Chaudhry, Nagarathnamma; Barnes, Gregory N.; Filbin, Marie T.; Carter, Bruce D.

    2010-01-01

    Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including Myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects cortical neurons. Here, we demonstrate that cortical neurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAG’s inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or over expression of activated AKT in cortical neurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis. PMID:20869442

  6. Role of glutamate in the regulation of the outgrowth and motility of neurites from mouse spinal cord neurons in culture

    PubMed Central

    OWEN, ALUN D.; BIRD, MARGARET M.

    1997-01-01

    The excitatory amino acid glutamate has been shown to be toxic to a number of neuronal cell types both in vitro and in vivo. It has also been shown to be capable of controlling the development of neurons grown in vitro. Using time-lapse video microscopy techniques the effects of glutamate on the rate of neurite outgrowth and growth cone motility were examined on cultured mouse spinal cord neurons. Concentrations in the range of 1 to 100 µ M caused a significant inhibition of neurite outgrowth and concentrations of 10 and 100 µ M significantly inhibited growth cone activity. In addition it was shown that the kainate/AMPA receptor antagonist (±)3-(2-carbvoxypiperazin-4-yl)-propyl-l-phosphonic acid, but not the NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione, was capable of blocking the inhibitory actions of glutamate on both outgrowth and motility. These results show that, at least in the culture system employed, glutamate might have a role in regulating neuronal development and function. PMID:9306206

  7. Increasing tPA Activity in Astrocytes Induced by Multipotent Mesenchymal Stromal Cells Facilitate Neurite Outgrowth after Stroke in the Mouse

    PubMed Central

    Xin, Hongqi; Li, Yi; Shen, Li Hong; Liu, Xianshuang; Wang, Xinli; Zhang, Jing; Pourabdollah-Nejad D, Siamak; Zhang, Chunling; Zhang, Li; Jiang, Hao; Zhang, Zheng Gang; Chopp, Michael

    2010-01-01

    We demonstrate that tissue plasminogen activator (tPA) and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS) after treatment of stroke with multipotent mesenchymal stromal cells (MSCs). In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo) significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ) compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1) expression in astrocytes under normal and oxygen and glucose deprivation (OGD) conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke. PMID:20140248

  8. PACAP induces neurite outgrowth in cultured trigeminal ganglion cells and recovery of corneal sensitivity after flap surgery in rabbits.

    PubMed

    Fukiage, Chiho; Nakajima, Takeshi; Takayama, Yoshiko; Minagawa, Yoko; Shearer, Thomas R; Azuma, Mitsuyoshi

    2007-02-01

    To evaluate the ability of pituitary adenylate cyclase-activating polypeptide (PACAP) to induce growth of neuronal processes in cultured trigeminal ganglion cells, and to accelerate neurite outgrowth and recovery of corneal sensitivity after creation of a corneal flap in a rabbit model of laser-assisted in situ keratomileusis (LASIK) surgery. Animal study. The cDNA of rabbit PACAP was sequenced, and the expression of PACAP receptors in the trigeminal ganglia from rabbits was quantified by quantitative real-time polymerase chain reaction. Trigeminal ganglion cells were isolated from rabbits and cultured for 48 hours with or without PACAP27 (bioactive N-terminal peptide from PACAP). Cells were stained with antibody against neurofilaments, and neurite outgrowth was quantified by cell counting. In the rabbit LASIK model, a corneal flap with a planned thickness of 130 microm and 8.5 mm diameter was created with a microkeratome. The rabbits then received eyedrops containing PACAP27 four times a day for eight weeks, and corneal sensitivity was measured. Neurite outgrowth was assessed by staining histologic sections of the flap area for cholinesterase. The deduced amino acid sequence of PACAP in rabbit was identical to that of human. PACAP receptor, PAC1, was highly expressed in trigeminal ganglia from newborn and adult rabbits. PACAP27 at 1 microM induced growth of neuronal processes in cultured primary trigeminal ganglion cells. In the LASIK model, extensions of neuronal processes from amputated nerve trunks in cornea were observed after administration of eyedrops containing 1 or 10 microM PACAP27. The 10 microM PACAP27 treatment also greatly accelerated recovery of corneal sensitivity. PACAP may be a candidate drug for ameliorating dry eye after LASIK surgery.

  9. TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells

    PubMed Central

    2017-01-01

    Recycling endosomes are generally thought to play a central role in endocytic recycling, but recent evidence has indicated that they also participate in other cellular events, including cytokinesis, autophagy, and neurite outgrowth. Rab small GTPases are key regulators in membrane trafficking, and although several Rab isoforms, e.g., Rab11, have been shown to regulate recycling endosomal trafficking, the precise mechanism by which these Rabs regulate recycling endosomes is not fully understood. In this study, we focused on a Rab-GTPase-activating protein (Rab-GAP), one of the key regulators of Rabs, and comprehensively screened 43 mammalian Tre-2/Bub2/Cdc16 (TBC)/Rab-GAP-domain-containing proteins (TBC proteins) for proteins that specifically localize on recycling endosomes in mouse embryonic fibroblasts (MEFs). Four of the 43 mammalian TBC proteins screened, i.e., TBC1D11, TBC1D12, TBC1D14, and EVI5, were found to colocalize well with transferrin receptor, a well-known recycling endosome marker. We further investigated the biochemical properties of TBC1D12, a previously uncharacterized TBC protein. The results showed that TBC1D12 interacted with active Rab11 through its middle region and that it did not display Rab11-GAP activity in vitro. The recycling endosomal localization of TBC1D12 was found to depend on the expression of Rab11. We also found that TBC1D12 expression had no effect on common Rab11-dependent cellular events, e.g., transferrin recycling, in MEFs and that it promoted neurite outgrowth, a specialized Rab11-dependent cellular event, of PC12 cells independently of its GAP activity. These findings indicated that TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells. PMID:28384198

  10. Electrical Stimulation Using Conductive Polymer Polypyrrole Counters Reduced Neurite Outgrowth of Primary Prefrontal Cortical Neurons from NRG1-KO and DISC1-LI Mice.

    PubMed

    Zhang, Qingsheng; Esrafilzadeh, Dorna; Crook, Jeremy M; Kapsa, Robert; Stewart, Elise M; Tomaskovic-Crook, Eva; Wallace, Gordon G; Huang, Xu-Feng

    2017-02-15

    Deficits in neurite outgrowth, possibly involving dysregulation of risk genes neuregulin-1 (NRG1) and disrupted in schizophrenia 1 (DISC1) have been implicated in psychiatric disorders including schizophrenia. Electrical stimulation using conductive polymers has been shown to stimulate neurite outgrowth of differentiating human neural stem cells. This study investigated the use of the electroactive conductive polymer polypyrrole (Ppy) to counter impaired neurite outgrowth of primary pre-frontal cortical (PFC) neurons from NRG1-knock out (NRG1-KO) and DISC1-locus impairment (DISC1-LI) mice. Whereas NRG1-KO and DISC1-LI exhibited reduced neurite length and number of neurite branches compared to wild-type controls, this was not apparent for cultures on electroactive Ppy. Additionally, the use of the Ppy substrate normalised the synaptophysin and PSD95 protein and mRNA expression whereas both are usually reduced by NRG1-KO or DISC1-LI. Our findings support the utility of Ppy mediated electrical stimulation to prevent the reduction of neurite outgrowth and related synaptic protein expression in the primary PFC neurons from NRG1-KO and DISC1-LI mice, providing proof-of-concept for treating neurodevelopmental diseases including schizophrenia.

  11. Regulation of neurite outgrowth mediated by neuronal calcium sensor-1 and inositol 1,4,5-trisphosphate receptor in nerve growth cones.

    PubMed

    Iketani, M; Imaizumi, C; Nakamura, F; Jeromin, A; Mikoshiba, K; Goshima, Y; Takei, K

    2009-07-07

    Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1). The pharmacological inhibition of InsP(3) receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP(3)R1, which may play an important role in the regulation of neurite outgrowth.

  12. Electrical Stimulation Using Conductive Polymer Polypyrrole Counters Reduced Neurite Outgrowth of Primary Prefrontal Cortical Neurons from NRG1-KO and DISC1-LI Mice

    PubMed Central

    Zhang, Qingsheng; Esrafilzadeh, Dorna; Crook, Jeremy M.; Kapsa, Robert; Stewart, Elise M.; Tomaskovic-Crook, Eva; Wallace, Gordon G.; Huang, Xu-Feng

    2017-01-01

    Deficits in neurite outgrowth, possibly involving dysregulation of risk genes neuregulin-1 (NRG1) and disrupted in schizophrenia 1 (DISC1) have been implicated in psychiatric disorders including schizophrenia. Electrical stimulation using conductive polymers has been shown to stimulate neurite outgrowth of differentiating human neural stem cells. This study investigated the use of the electroactive conductive polymer polypyrrole (Ppy) to counter impaired neurite outgrowth of primary pre-frontal cortical (PFC) neurons from NRG1-knock out (NRG1-KO) and DISC1-locus impairment (DISC1-LI) mice. Whereas NRG1-KO and DISC1-LI exhibited reduced neurite length and number of neurite branches compared to wild-type controls, this was not apparent for cultures on electroactive Ppy. Additionally, the use of the Ppy substrate normalised the synaptophysin and PSD95 protein and mRNA expression whereas both are usually reduced by NRG1-KO or DISC1-LI. Our findings support the utility of Ppy mediated electrical stimulation to prevent the reduction of neurite outgrowth and related synaptic protein expression in the primary PFC neurons from NRG1-KO and DISC1-LI mice, providing proof-of-concept for treating neurodevelopmental diseases including schizophrenia. PMID:28198409

  13. The promotive effects of thymosin beta4 on neuronal survival and neurite outgrowth by upregulating L1 expression.

    PubMed

    Yang, Hao; Cheng, Xipeng; Yao, Qing; Li, Jingwen; Ju, Gong

    2008-11-01

    Thymosin beta(4) (Tbeta4) is a major actin-sequestering peptide widely distributed in mammalian tissues including the nervous system. The presence of this peptide in the nervous system likely plays a role in synaptogensis, axon growth, cell migration, and plastic changes in dendritic spine. However, the effects of Tbeta4 on the survival of neurons and axonal outgrowth have still not been fully understood. So far it is not clear if the effects of Tbeta4 are associated with L1 functions. In the present study, we hypothesized that Tbeta4-induced up-regulation of L1 synthesis could be involved in the survival and axon outgrowth of cultured spinal cord neurons. To test this hypothesis, primarily cultured neurons were prepared from the mouse spinal cord and treated with various concentrations of Tbeta4 ranging from 0.1 to 10 microg/ml. The analysis of L1 mRNA expression and protein synthesis in neurons was then carried out using RT-PCR and western blot assays, respectively. After the addition of Tbeta4 to cultures, cells were then treated with antibodies against distinct domains of L1-Fc. Subsequently, beta-tubulin III and L1 double-labeled indirect immunofluorescence was carried out. Meanwhile, L1 immunofluorescent reactivity was analyzed and compared in cells treated with Tbeta4. Furthermore, the number of beta-tubulin III-positive cells and neurite lengths were measured. We found that Tbeta4 enhanced L1 expression in a dose-dependent manner, and the highest L1 mRNA and protein synthesis in cells increased by more than 2.1- and 2.3-fold in the presence of Tbeta4 at identical concentrations, respectively. Moreover, it also dose dependently enhanced neurite outgrowth and neuronal survival. Compared to conditions without Tbeta4, the length of neurite and neuronal survival increased markedly in presence of 0.5, 1, and 5 microg/ml Tbeta4, respectively, whereas the effects of Tbeta4 were significantly attenuated or inhibited in the process of L1-Fc antibodies treatment

  14. Neurite outgrowth of NG108-15 cells induced by heat shock protein 90 inhibitors.

    PubMed

    Jin, Erika; Sano, Mamoru

    2008-12-01

    We previously reported that radicicol (Rad) and geldanamycin (Geld), heat shock protein 90 (Hsp90) inhibitors, potentiate neurite growth of cultured sensory neurons from chick embryo. We now show that the antibiotics induce neurite growth in NG108-15 cells. Treatment of the cells with these drugs caused transient decrease in protein levels of Raf1, ERK1/2, phosphorylated ERK1/2, Akt1, and CDK4. The neurite growth of NG108-15 induced by the inhibitors was blocked by actynomycin D, but the neurite growth stimulated by dbcAMP in the cells was not affected. The neurite growth could be due to a change in the synthesis of some specific protein(s) and is speculated to be due to the transient downregulation of particular-signaling molecules stabilized by Hsp90.

  15. Sensitivity of Neural Stem Cell Survival, Differentiation and Neurite Outgrowth within 3D Hydrogels to Environmental Heavy Metals

    PubMed Central

    Tasneem, Sameera; Farrell, Kurt; Lee, Moo-Yeal; Kothapalli, Chandrasekhar R.

    2015-01-01

    We investigated the sensitivity of embryonic murine neural stem cells exposed to 10 pM – 10 μM concentrations of three heavy metals (Cd, Hg, Pb), continuously for 14 days within 3D collagen hydrogels. Critical endpoints for neurogenesis such as survival, differentiation and neurite outgrowth were assessed. Results suggest significant compromise in cell viability within the first four days at concentrations ≥ 10 nM, while lower concentrations induced a more delayed effect. Mercury and lead suppressed neural differentiation at as low as 10 pM concentration within 7 days, while all three metals inhibited neural and glial differentiation by day 14. Neurite outgrowth remained unaffected at lower cadmium or mercury concentrations (≤ 100 pM), but was completely repressed beyond day 1 at higher concentrations. Higher metal concentrations (≥ 100 pM) suppressed NSC differentiation to motor or dopaminergic neurons. Cytokines and chemokines released by NSCs, and the sub-cellular mechanisms by which metals induce damage to NSCs have been quantified and correlated to phenotypic data. The observed degree of toxicity in NSC cultures is in the order: lead > mercury > cadmium. Results point to the use of biomimetic 3D culture models to screen the toxic effects of heavy metals during developmental stages, and investigate their underlying mechanistic pathways. PMID:26621541

  16. 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

  17. Involvement of vimentin in neurite outgrowth damage induced by fipronil in SH-SY5Y cells.

    PubMed

    Ruangjaroon, Theetat; Chokchaichamnankit, Daranee; Srisomsap, Chantragan; Svasti, Jisnuson; Paricharttanakul, N Monique

    2017-05-06

    Fipronil, a phenylpyrazole insecticide, is more selective in its potency towards insects than humans and is thus commonly used. In this study, we demonstrated that exposure to fipronil may pose a human health risk. We observed in vitro the shortening of neurite outgrowths of SH-SY5Y neuroblastoma cells upon treatment with fipronil, even at a non-cytotoxic concentration. Fipronil induced apoptosis involving caspase-6, which is an apoptotic effector highly implicated in neurodegenerative diseases. Moreover, at a concentration that did not induce apoptosis, mitochondrial dysfunction and autophagic vacuole formation were detected. Interestingly using proteomics, we identified vimentin to be dramatically expressed by SH-SY5Y cells as a response to fipronil treatment. Not only did the expression of total vimentin increase, different isoforms were observed, indicating alterations in post-translational modifications. Vimentin was localized at the neurite outgrowth, possibly to repair the damage in cellular structure. However at high concentrations of fipronil, vimentin was found in less defined fibrils, in bridge-like formation, and dense surrounding vacuoles. In all, our results indicate that vimentin plays an important role in fipronil-induced neurotoxicity in SH-SY5Y cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. 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

    2016-04-08

    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.

  19. Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress: Involving of mitochondria and energy metabolism.

    PubMed

    He, Weiliang; Cui, Lili; Zhang, Cong; Zhang, Xiangjian; He, Junna; Xie, Yanzhao; Chen, Yanxia

    2017-01-01

    Oxidative stress has been demonstrated to be involved in the etiology of several neurobiological disorders. Sonic hedgehog (Shh), a secreted glycoprotein factor, has been implicated in promoting several aspects of brain remodeling process. Mitochondria may play an important role in controlling fundamental processes in neuroplasticity. However, little evidence is available about the effect and the potential mechanism of Shh on neurite outgrowth in primary cortical neurons under oxidative stress. Here, we revealed that Shh treatment significantly increased the viability of cortical neurons in a dose-dependent manner, which was damaged by hydrogen peroxide (H2O2). Shh alleviated the apoptosis rate of H2O2-induced neurons. Shh also increased neuritogenesis injuried by H2O2 in primary cortical neurons. Moreover, Shh reduced the generation of reactive oxygen species (ROS), increased the activities of SOD and and decreased the productions of MDA. In addition, Shh protected mitochondrial functions, elevated the cellular ATP levels and amelioratesd the impairment of mitochondrial complex II activities of cortical neurons induced by H2O2. In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H2O2 -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress.

  20. Sensitivity of neural stem cell survival, differentiation and neurite outgrowth within 3D hydrogels to environmental heavy metals.

    PubMed

    Tasneem, Sameera; Farrell, Kurt; Lee, Moo-Yeal; Kothapalli, Chandrasekhar R

    2016-02-03

    We investigated the sensitivity of embryonic murine neural stem cells exposed to 10 pM-10 μM concentrations of three heavy metals (Cd, Hg, Pb), continuously for 14 days within 3D collagen hydrogels. Critical endpoints for neurogenesis such as survival, differentiation and neurite outgrowth were assessed. Results suggest significant compromise in cell viability within the first four days at concentrations ≥10 nM, while lower concentrations induced a more delayed effect. Mercury and lead suppressed neural differentiation at as low as 10 pM concentration within 7 days, while all three metals inhibited neural and glial differentiation by day 14. Neurite outgrowth remained unaffected at lower cadmium or mercury concentrations (≤100 pM), but was completely repressed beyond day 1 at higher concentrations. Higher metal concentrations (≥100 pM) suppressed NSC differentiation to motor or dopaminergic neurons. Cytokines and chemokines released by NSCs, and the sub-cellular mechanisms by which metals induce damage to NSCs have been quantified and correlated to phenotypic data. The observed degree of toxicity in NSC cultures is in the order: lead>mercury>cadmium. Results point to the use of biomimetic 3D culture models to screen the toxic effects of heavy metals during developmental stages, and investigate their underlying mechanistic pathways.

  1. Neurotrophic effects of GnRH on neurite outgrowth and neurofilament protein expression in cultured cerebral cortical neurons of rat embryos.

    PubMed

    Quintanar, J Luis; Salinas, Eva

    2008-06-01

    The presence of GnRH receptor in cerebral cortical neurons of rat embryos and adult rats has been described. In this work, we studied the effects of GnRH on outgrowth and length of neurites and cytoskeletal neurofilament proteins expression (NF-68 and NF-200 kDa) by immunoblot of cultured cerebral cortical neurons of rat embryos. Our results show that GnRH increases both outgrowth and length of neurites accompanied by an increase in neurofilaments expression. It is conceivable that GnRH plays a role in neuronal plasticity parallel to its gonadal function.

  2. cAMP response element-binding protein and Yes-associated protein form a feedback loop that promotes neurite outgrowth.

    PubMed

    Chen, Lei; Feng, Peimin; Peng, Anjiao; Qiu, Xiangmiao; Zhu, Xi; He, Shixu; Zhou, Dong

    2017-08-31

    The cAMP response element-binding (CREB) protein is a member of the CREB/activating transcription factor family that is activated by various extracellular stimuli. It has been shown that CREB-dependent transcription stimulation plays a key role in neuronal differentiation and plasticity, but the underlying mechanisms remain largely elusive. Here, we show that Yes-associated protein (YAP) is a direct target induced by CREB upon retinoic acid (RA)-induced neurite outgrowth stimuli in N2a cells. Interestingly, YAP knockout using the CRISPR/Cas9 system inhibits neuronal differentiation and reduced neurite length. We further show that YAP could directly bind to CREB via its N-terminal region, and loss of YAP results in instability of phosphorylated CREB upon neurite outgrowth stimuli. Transient expression of YAP could largely restore CREB expression and neurite outgrowth in YAP knockout cells. Together, our results suggest that CREB and YAP form a positive feedback loop that is critical to maintain the stability of phosphorylated CREB and promote neurite outgrowth. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  3. c-Jun Amino-Terminal Kinase is Involved in Valproic Acid-Mediated Neuronal Differentiation of Mouse Embryonic NSCs and Neurite Outgrowth of NSC-Derived Neurons.

    PubMed

    Lu, Lu; Zhou, Hengxing; Pan, Bin; Li, Xueying; Fu, Zheng; Liu, Jun; Shi, Zhongju; Chu, Tianci; Wei, Zhijian; Ning, Guangzhi; Feng, Shiqing

    2017-04-01

    Valproic acid (VPA), an anticonvulsant and mood-stabilizing drug, can induce neuronal differentiation, promote neurite extension and exert a neuroprotective effect in central nervous system (CNS) injuries; however, comparatively little is known regarding its action on mouse embryonic neural stem cells (NSCs) and the underlying molecular mechanism. Recent studies suggested that c-Jun N-terminal kinase (JNK) is required for neurite outgrowth and neuronal differentiation during neuronal development. In the present study, we cultured mouse embryonic NSCs and treated the cells with 1 mM VPA for up to 7 days. The results indicate that VPA promotes the neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSC-derived neurons; moreover, VPA induces the phosphorylation of c-Jun by JNK. In contrast, the specific JNK inhibitor SP600125 decreased the VPA-stimulated increase in neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSC-derived neurons. Taken together, these results suggest that VPA promotes neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSC-derived neurons. Moreover, JNK activation is involved in the effects of VPA stimulation.

  4. 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

  5. Cellular form of prion protein inhibits Reelin-mediated shedding of Caspr from the neuronal cell surface to potentiate Caspr-mediated inhibition of neurite outgrowth.

    PubMed

    Devanathan, Vasudharani; Jakovcevski, Igor; Santuccione, Antonella; Li, Shen; Lee, Hyun Joon; Peles, Elior; Leshchyns'ka, Iryna; Sytnyk, Vladimir; Schachner, Melitta

    2010-07-07

    Extension of axonal and dendritic processes in the CNS is tightly regulated by outgrowth-promoting and -inhibitory cues to assure precision of synaptic connections. We identify a novel role for contactin-associated protein (Caspr) as an inhibitory cue that reduces neurite outgrowth from CNS neurons. We show that proteolysis of Caspr at the cell surface is regulated by the cellular form of prion protein (PrP), which directly binds to Caspr. PrP inhibits Reelin-mediated shedding of Caspr from the cell surface, thereby increasing surface levels of Caspr and potentiating the inhibitory effect of Caspr on neurite outgrowth. PrP deficiency results in reduced levels of Caspr at the cell surface, enhanced neurite outgrowth in vitro, and more efficient regeneration of axons in vivo following spinal cord injury. Thus, we reveal a previously unrecognized role for Caspr and PrP in inhibitory modulation of neurite outgrowth in CNS neurons, which is counterbalanced by the proteolytic activity of Reelin.

  6. Impaired neurogenesis and neurite outgrowth in an HIV-gp120 transgenic model is reversed by exercise via BDNF production and Cdk5 regulation

    PubMed Central

    Lee, Myoung-Hwa; Amin, Niranjana D.; Venkatesan, Arun; Wang, Tongguang; Tyagi, Richa; Pant, Harish C.; Nath, Avindra

    2013-01-01

    Human immunodeficiency virus (HIV) infection associated neurocognitive disorders (HAND) is accompanied with brain atrophy. In these patients, impairment of adult neurogenesis and neurite outgrowth in the hippocampus may contribute to the cognitive dysfunction. Although running exercises can enhance neurogenesis and normalize neurite outgrowth, the underlying molecular mechanisms are not well understood. The HIV envelope protein, gp120, has been shown to impair neurogenesis. Using a gp120 transgenic mouse model, we demonstrate that exercise stimulated neural progenitor cell (NPC) proliferation in the hippocampal dentate gyrus and increased the survival rate and generation of newborn cells. However sustained exercise activity was necessary since the effects were reversed by detraining. Exercise also normalized dendritic outgrowth of neurons. Furthermore, it also increased the expression of hippocampal brainderived neurotrophic factor (BDNF) and normalized hyperactivation of cyclin-dependent kinase 5 (Cdk5). Hyper-activated Cdk5 or gp120 treatment led to aberrant neurite outgrowth and BDNF treatment normalized the neurite outgrowth in NPC cultures. These results suggest that sustained exercise has trophic activity on the neuronal lineage which is mediated by Cdk5 modulation of the BDNF pathway. PMID:23982957

  7. 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

  8. 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.

  9. Effect of 710 nm visible light irradiation on neurite outgrowth in primary rat cortical neurons following ischemic insult.

    PubMed

    Choi, Dong-Hee; Lee, Kyoung-Hee; Kim, Ji-Hye; Kim, Moon Young; Lim, Jeong Hoon; Lee, Jongmin

    2012-06-01

    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. 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(2) and 50 mW/cm(2)) were given once to four times within 8h at 2h 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. 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 increased with LED treatment in both OGD-exposed and normal cells (p<0.05). Our data suggest that LED treatment may promote synaptogenesis through MAPK activation and subsequently protect cell death in the in vitro stroke model. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Comparison of the Effects of Curcumin and RG108 on NGF-Induced PC-12 Adh Cell Differentiation and Neurite Outgrowth.

    PubMed

    Dikmen, Miriş

    2017-04-01

    DNA methyltransferases (DNMTs) are promising epigenetic targets for the development of novel drugs, especially for neurodegenerative disorders. In recent years, there has been increased interest in small molecules that can cross the blood-brain barrier for the treatment of neurodegenerative diseases. Therefore, comparing the neuronal differentiative effects of a natural compound curcumin and a synthetic small molecule RG108 was the aim of this study. The effects of curcumin and RG108 on neuronal differentiation and neurite outgrowth were investigated in the PC-12 Adh cell line. First, a nontoxic concentration was determined to be 100 nM with WST-1 assay. Subsequently, cells were treated with 100 nM curcumin and RG108 alone or in combination with 50 nM nerve growth factor (NGF). Cell differentiations were evaluated by a real-time cell analyzer system. Neurite outgrowth was determined and morphologically shown by immunofluorescence staining with anti-beta III tubulin antibody on PC-12 Adh cells. Also, growth-associated protein-43 (GAP-43) and β-tubulin III mRNA expression levels, associated with neurite outgrowth promotion, were determined with real-time polymerase chain reaction (RT-PCR). According to our results, 100 nM curcumin and RG108 significantly induced neurite outgrowth of PC-12 Adh cells with 50 nM NGF. Curcumin + NGF combination further increased cell differentiations and total neurite lengths more than curcumin alone and RG108 + NGF combination groups. Strikingly, curcumin and NGF combination upregulated GAP-43 and β-tubulin mRNA expression levels excessively. In conclusion, curcumin was found to be more effective than RG108 on neuronal differentiation and neurite outgrowth of PC-12 Adh cells in a combination with NGF. Therefore, natural DNMT1 inhibitors, such as curcumin, can be a novel approach for the neurodegenerative disorders treatment.

  11. FK962 induces neurite outgrowth in cultured monkey trigeminal ganglion cells.

    PubMed

    Nakajima, Emi; Walkup, Ryan D; Shearer, Thomas R; Azuma, Mitsuyoshi

    2017-01-01

    Corneal sensation, cell proliferation, and wound healing all depend on adequate corneal innervation. Disruption of corneal innervation can lead to dry eye and delayed wound healing. Our studies in rats and rabbits show that the substituted fluorobenzamide drug FK962 accelerates the extension of neuronal processes and recovery of corneal sensitivity. The purpose of the present study was 1) to determine whether FK962 induces sprouting and elongation of neurites in cultured monkey trigeminal ganglion cells, and 2) to investigate the involvement of the neurotrophic peptide GDNF in FK962-induced neurite elongation. Dissociated, cultured trigeminal ganglion cells, containing neuronal and Schwann cells were cultured for 48 h with or without FK962. Neuronal elongation was evaluated by immunostaining with a neurofilament-specific antibody. Culture with or without GDNF, or with antibody against GDNF, was used to determine the role of GDNF in FK962-induced neurite elongation. FK962 or GDNF were found to significantly induce neurite elongation. The GDNF antibody significantly inhibited elongation induced by FK962. GDNF was found to be a mediator of FK962-induced neurite elongation in a relevant primate model. FK962 may be a candidate drug for treatment of neurotrophic disorders in the human cornea.

  12. Minocycline Promotes Neurite Outgrowth of PC12 Cells Exposed to Oxygen-Glucose Deprivation and Reoxygenation Through Regulation of MLCP/MLC Signaling Pathways.

    PubMed

    Tao, Tao; Feng, Jin-Zhou; Xu, Guang-Hui; Fu, Jie; Li, Xiao-Gang; Qin, Xin-Yue

    2017-04-01

    Minocycline, a semi-synthetic second-generation derivative of tetracycline, has been reported to exert neuroprotective effects both in animal models and in clinic trials of neurological diseases. In the present study, we first investigated the protective effects of minocycline on oxygen-glucose deprivation and reoxygenation-induced impairment of neurite outgrowth and its potential mechanism in the neuronal cell line, PC12 cells. We found that minocycline significantly increased cell viability, promoted neurite outgrowth and enhanced the expression of growth-associated protein-43 (GAP-43) in PC12 cells exposed to oxygen-glucose deprivation/reoxygenation injury. In addition, immunoblots revealed that minocycline reversed the overexpression of phosphorylated myosin light chain (MLC) and the suppression of activated extracellular signal-regulated kinase 1/2 (ERK1/2) caused by oxygen-glucose deprivation/reoxygenation injury. Moreover, the minocycline-induced neurite outgrowth was significantly blocked by Calyculin A (1 nM), an inhibitor of myosin light chain phosphatase (MLCP), but not by an ERK1/2 inhibitor (U0126; 10 μM). These findings suggested that minocycline activated the MLCP/MLC signaling pathway in PC12 cells after oxygen-glucose deprivation/reoxygenation injury, which resulted in the promotion of neurite outgrowth.

  13. Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro.

    PubMed

    Martens, Wendy; Sanen, Kathleen; Georgiou, Melanie; Struys, Tom; Bronckaers, Annelies; Ameloot, Marcel; Phillips, James; Lambrichts, Ivo

    2014-04-01

    In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d-hDPSCs), which promoted survival and neurite outgrowth in 2-dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d-hDPSCs in a 3-dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d-hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell-based therapies as treatment for peripheral nerve injury.

  14. 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

  15. Potentiation of Nerve Growth Factor-Induced Neurite Outgrowth by Fluvoxamine: Role of Sigma-1 Receptors, IP3 Receptors and Cellular Signaling Pathways

    PubMed Central

    Nishimura, Tomoko; Ishima, Tamaki; Iyo, Masaomi; Hashimoto, Kenji

    2008-01-01

    Background Selective serotonin reuptake inhibitors (SSRIs) have been widely used and are a major therapeutic advance in psychopharmacology. However, their pharmacology is quite heterogeneous. The SSRI fluvoxamine, with sigma-1 receptor agonism, is shown to potentiate nerve-growth factor (NGF)-induced neurite outgrowth in PC 12 cells. However, the precise cellular and molecular mechanisms underlying potentiation by fluvoxamine are not fully understood. In this study, we examined the roles of cellular signaling pathways in the potentiation of NGF-induced neurite outgrowth by fluvoxamine and sigma-1 receptor agonists. Methods and Findings The effects of three SSRIs (fluvoxamine, sertraline, paroxetine) and three sigma-1 receptor agonists (SA4503, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP), and dehydroepiandrosterone (DHEA)-sulfate) on NGF-induced neurite outgrowth in PC12 cells were examined. Also examined were the effects of the sigma-1 receptor antagonist NE-100, inositol 1,4,5-triphosphate (IP3) receptor antagonist, and specific inhibitors of signaling pathways in the potentiation of NGF-induced neurite outgrowth by selective sigma-1 receptor agonist SA4503. Fluvoxamine (but not sertraline or paroxetine) and the sigma-1 receptor agonists SA4503, PPBP, and DHEA-sulfate significantly potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. The potentiation by fluvoxamine and the three sigma-1 receptor agonists was blocked by co-administration of the selective sigma-1 receptor antagonist NE-100, suggesting that sigma-1 receptors play a role in blocking the enhancement of NGF-induced neurite outgrowth. Moreover, the potentiation by SA4503 was blocked by co-administration of the IP3 receptor antagonist xestospongin C. In addition, the specific inhibitors of phospholipase C (PLC-γ), phosphatidylinositol 3-kinase (PI3K), p38MAPK, c-Jun N-terminal kinase (JNK), and the Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways

  16. P2X1 Receptor-Mediated Ca(2+) Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

    PubMed

    Back, Moon Jung; Lee, Hae Kyung; Lee, Joo Hyun; Fu, Zhicheng; Son, Mi Won; Choi, Sang Zin; Go, Hyo Sang; Yoo, Sungjae; Hwang, Sun Wook; Kim, Dae Kyong

    2016-11-16

    Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

  17. The Akt-nitric oxide-cGMP pathway contributes to nerve growth factor-mediated neurite outgrowth in apolipoprotein E knockout mice.

    PubMed

    Hashikawa-Hobara, Narumi; Hashikawa, Naoya; Yutani, Chikao; Zamami, Yoshito; Jin, Xin; Takatori, Shingo; Mio, Mitsunobu; Kawasaki, Hiromu

    2011-08-01

    Apolipoprotein E (apo)-deficient [apoE(-/-)] mice have peripheral sensory nerve defects and a reduced and delayed response to noxious thermal stimuli. However, to date, no report has focused on the influence of apoE deficiency on calcitonin gene-related peptide (CGRP)-containing nerve fiber extensions. We have shown that the density of CGRP-containing nerve fibers decreases in mesenteric arteries of apoE(-/-) mice compared with wild-type mice. Here, we investigated whether apoE deficiency is involved in nerve growth factor (NGF)-induced CGRP-containing nerve regeneration using apoE(-/-) mice. NGF-mediated CGRP-like immunoreactivity (LI)-neurite outgrowth in apoE(-/-) cultured dorsal root ganglia (DRG) cells was significantly lower than that in wild-type cultures. However, the level of NGF receptor mRNA in apoE(-/-) DRG cells was similar to that in wild-type mice. To clarify the mechanism of the impaired ability of NGF-mediated neurite outgrowth, we focused on the Akt-nitric oxide (NO)-cGMP pathway. Expression of phosphorylated Akt was significantly reduced in apoE(-/-) DRG. The NO donor, sodium nitroprusside or S-nitroso-N-acetylpenicillamine, did not affect NGF-mediated neurite outgrowth in apoE(-/-) cultured DRG cells. However, 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt n-hydrate, a cGMP analog, induced NGF-mediated nerve facilitation similar to wild-type NGF-mediated neurite outgrowth levels. Furthermore, in apoE(-/-) DRG, soluble guanylate cyclase expression was significantly lower than that in wild-type DRG. These results suggest that in apoE(-/-) mice the Akt-NO-cGMP pathway is impaired, which may be caused by NGF-mediated CGRP-LI-neurite outgrowth defects.

  18. The adaptor protein SH2B3 (Lnk) negatively regulates neurite outgrowth of PC12 cells and cortical neurons.

    PubMed

    Wang, Tien-Cheng; Chiu, Hsun; Chang, Yu-Jung; Hsu, Tai-Yu; Chiu, Ing-Ming; Chen, Linyi

    2011-01-01

    SH2B adaptor protein family members (SH2B1-3) regulate various physiological responses through affecting signaling, gene expression, and cell adhesion. SH2B1 and SH2B2 were reported to enhance nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells, a well-established neuronal model system. In contrast, SH2B3 was reported to inhibit cell proliferation during the development of immune system. No study so far addresses the role of SH2B3 in the nervous system. In this study, we provide evidence suggesting that SH2B3 is expressed in the cortex of embryonic rat brain. Overexpression of SH2B3 not only inhibits NGF-induced differentiation of PC12 cells but also reduces neurite outgrowth of primary cortical neurons. SH2B3 does so by repressing NGF-induced activation of PLCγ, MEK-ERK1/2 and PI3K-AKT pathways and the expression of Egr-1. SH2B3 is capable of binding to phosphorylated NGF receptor, TrkA, as well as SH2B1β. Our data further demonstrate that overexpression of SH2B3 reduces the interaction between SH2B1β and TrkA. Consistent with this finding, overexpressing the SH2 domain of SH2B3 is sufficient to inhibit NGF-induced neurite outgrowth. Together, our data demonstrate that SH2B3, unlike the other two family members, inhibits neuronal differentiation of PC12 cells and primary cortical neurons. Its inhibitory mechanism is likely through the competition of TrkA binding with the positive-acting SH2B1 and SH2B2.

  19. Arginyltransferase ATE1 is targeted to the neuronal growth cones and regulates neurite outgrowth during brain development.

    PubMed

    Wang, Junling; Pavlyk, Iuliia; Vedula, Pavan; Sterling, Stephanie; Leu, N Adrian; Dong, Dawei W; Kashina, Anna

    2017-10-01

    Arginylation is an emerging protein modification mediated by arginyltransferase ATE1, shown to regulate embryogenesis and actin cytoskeleton, however its functions in different physiological systems are not well understood. Here we analyzed the role of ATE1 in brain development and neuronal growth by producing a conditional mouse knockout with Ate1 deletion in the nervous system driven by Nestin promoter (Nes-Ate1 mice). These mice were weaker than wild type, resulting in low postnatal survival rates, and had abnormalities in the brain that suggested defects in neuronal migration. Cultured Ate1 knockout neurons showed a reduction in the neurite outgrowth and the levels of doublecortin and F-actin in the growth cones. In wild type, ATE1 prominently localized to the growth cones, in addition to the cell bodies. Examination of the Ate1 mRNA sequence reveals the existence of putative zipcode-binding sequences involved in mRNA targeting to the cell periphery and local translation at the growth cones. Fluorescence in situ hybridization showed that Ate1 mRNA localized to the tips of the growth cones, likely due to zipcode-mediated targeting, and this localization coincided with spots of localization of arginylated β-actin, which disappeared in the presence of protein synthesis inhibitors. We propose that zipcode-mediated co-targeting of Ate1 and β-actin mRNA leads to localized co-translational arginylation of β-actin that drives the growth cone migration and neurite outgrowth. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. 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.

  1. Thrombospondin-4, an extracellular matrix protein expressed in the developing and adult nervous system promotes neurite outgrowth

    PubMed Central

    1995-01-01

    Extracellular matrix (ECM) molecules are involved in multiple aspects of cell-to-cell signaling during development and in the adult. In nervous system development, specific recognition processes, e.g., during axonal pathfinding and synaptogenesis involve modulation and signaling by ECM components. Much less is known about their presence and possible roles in the adult nervous system. We now report that thrombospondin-4 (TSP-4), a recently discovered member of the TSP gene family is expressed by neurons, promotes neurite outgrowth, and accumulates at the neuromuscular junction and at certain synapse-rich structures in the adult. To search for muscle genes that may be involved in neuromuscular signaling, we isolated cDNAs induced in adult skeletal muscle by denervation. One of these cDNAs coded for the rat homologue of TSP-4. In skeletal muscle, it was expressed by muscle interstitial cells. The transcript was further detected in heart and in the developing and adult nervous system, where it was expressed by a wide range of neurons. An antiserum to the unique carboxyl-terminal end of the protein allowed to specifically detect TSP-4 in transfected cells in vitro and on cryostat sections in situ. TSP-4 associated with ECM structures in vitro and in vivo. In the adult, it accumulated at the neuromuscular junction and at synapse-rich structures in the cerebellum and retina. To analyze possible activities of TSP-4 towards neurons, we carried out coculture experiments with stably transfected COS cells and motor, sensory, or retina neurons. These experiments revealed that TSP-4 was a preferred substrate for these neurons, and promoted neurite outgrowth. The results establish TSP-4 as a neuronal ECM protein associated with certain synapse-rich structures in the adult. Its activity towards embryonic neurons in vitro and its distribution in vivo suggest that it may be involved in local signaling in the developing and adult nervous system. PMID:7490284

  2. The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by beta-amyloid peptide.

    PubMed

    Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, W E

    2010-05-01

    beta-Amyloid peptide (Abeta) 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. We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Abeta-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Abeta and on neurite outgrowth in PC12 cells were investigated. Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Abeta(1-42). Similar protective effects against Abeta(1-42) 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 Abeta load was markedly diminished in the brain of those animals after treatment with piracetam. Abeta 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 Abeta-induced mitochondrial dysfunction and Abeta-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Abeta on brain function.

  3. Upregulated Expression of TRIM32 Is Involved in Schwann Cell Differentiation, Migration and Neurite Outgrowth After Sciatic Nerve Crush.

    PubMed

    Liu, Yonghua; Wu, Weijie; Yang, Huiguang; Zhou, Zhengming; Zhu, Xiaojian; Sun, Chi; Liu, Yuxi; Yu, Zhaohui; Chen, Yuyan; Wang, Youhua

    2017-04-01

    Tripartite motif containing 32 (TRIM32), a member of the tripartite motif (TRIM) family, plays an indispensable role in myoblast proliferation. It also regulates neuron and skeletal muscle stem cell differentiation. Although it is of great importance, we know little about the roles of TRIM32 during peripheral nervous system injury. Here, we examined the dynamic changes of TRIM32 in acute sciatic nerve crush (SNC) model. After crush, TRIM32 rapidly increased and reached the climax at 1 week but then gradually declined to the normal level at 4 weeks post-injury. Meanwhile, we observed similar changes of Oct-6. What is more, we found co-localization of TRIM32 with S100 and Oct-6 in 1-week-injured tissues using double immunofluorescent staining. In further vitro experiments, enhancive expression of TRIM32 was detected during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation and nerve growth factor (NGF)-induced PC12 cell neurite outgrowth. More interestingly, specific si-TRIM32-transfected RSC96 cells exhibited obvious reduction in the ability of migration. Taken together, we inferred that upregulated TRIM32 was not only involved in the differentiation and migration of Schwann cells but the neurite elongation after SNC.

  4. 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.

  5. Enhancement of neurite outgrowth in neuronal-like cells following boron nitride nanotube-mediated stimulation.

    PubMed

    Ciofani, Gianni; Danti, Serena; D'Alessandro, Delfo; Ricotti, Leonardo; Moscato, Stefania; Bertoni, Giovanni; Falqui, Andrea; Berrettini, Stefano; Petrini, Mario; Mattoli, Virgilio; Menciassi, Arianna

    2010-10-26

    In this paper, we propose an absolutely innovative technique for the electrical stimulation of cells, based on piezoelectric nanoparticles. Ultrasounds are used to impart mechanical stress to boron nitride nanotubes incubated with neuronal-like PC12 cells. By virtue of their piezoelectric properties, these nanotubes can polarize and convey electrical stimuli to the cells. PC12 stimulated with the present method exhibit neurite sprout 30% greater than the control cultures after 9 days of treatment.

  6. Effects of avermectins on neurite outgrowth in differentiating mouse neuroblastoma N2a cells.

    PubMed

    Sun, Ying-Jian; Long, Ding-Xin; Li, Wei; Hou, Wei-Yuan; Wu, Yi-Jun; Shen, Jian-Zhong

    2010-02-01

    Avermectins (AVMs) are macrocyclic lactone compounds that have been widely used as parasiticides in veterinary and human medicine and as pesticides in agriculture and horticulture. The multidrug resistance transporter, P-glycoprotein (P-gp), is associated with the efflux transport of AVMs and other drugs across the blood-brain and placental barrier, and plays an important role in attenuating the neurotoxicity and developmental toxicity of AVMs. In this study, the mouse neuroblastoma N2a cell line was used to investigate the neurotoxicity of two AVM derivatives: abamectin (ABM) and doramectin (DOR). We found that both these compounds caused significant dose-dependent inhibition of neurite growth in differentiating N2a cells. In addition, Western blotting analysis showed that ABM and DOR significantly inhibited the expression of not only P-gp but also the cytoskeletal proteins, beta-actin and beta-tubulin. This suggests ABM and DOR may inhibit neurite growth by down-regulating the expression of P-gp and cytoskeletal proteins. Furthermore, knockdown of P-gp expression by RNA interference in N2a cells reduced neurite growth even in the absence of ABM and DOR, and reduced it even more in the presence of low levels of these compounds. These results suggest that even subcytotoxic levels of ABM and DOR can be neurotoxic in differentiating cells and that this neurotoxicity may, at least in part, be the result of the down-regulation of P-gp and cytoskeletal proteins.

  7. Valproic Acid Modifies Synaptic Structure and Accelerates Neurite Outgrowth Via the Glycogen Synthase Kinase-3β Signaling Pathway in an Alzheimer's Disease Model.

    PubMed

    Long, Zhi-Min; Zhao, Lei; Jiang, Rong; Wang, Ke-Jian; Luo, Shi-Fang; Zheng, Min; Li, Xiao-Feng; He, Gui-Qiong

    2015-11-01

    Tau hyperphosphorylation and amyloid β-peptide overproduction, caused by altered localization or abnormal activation of glycogen synthase kinase-3β (GSK-3β), is a pathogenic mechanism in Alzheimer's disease (AD). Valproic acid (VPA) attenuates senile plaques and neuronal loss. Here, we confirmed that VPA treatment improved spatial memory in amyloid precursor protein (APP)/presenilin 1 (PS 1) double-transgenic mice and investigated the effect of VPA on synaptic structure and neurite outgrowth. We used ultrastructural analysis, immunocytochemistry, immunofluorescence staining, and Western blot analysis to assess the effect of VPA treatment in mice. VPA treatment thickened the postsynaptic density, increased the number of presynaptic vesicles, and upregulated the expression of synaptic markers PSD-95 and GAP43. VPA increased neurite length of hippocampal neurons in vivo and in vitro. In VPA-treated AD mouse brain, inactivated GSK-3β (pSer9-GSK-3β) was markedly increased, while hyperphosphorylation of tau at Ser396 and Ser262 was decreased; total tau levels remained similar. VPA treatment notably improved pSer133-cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) levels, which are associated with synaptic function and neurite outgrowth. VPA improves behavioral deficits in AD, modifies synaptic structure, and accelerates neurite outgrowth, by inhibiting the activity of GSK-3β, decreasing hyperphosphorylated tau, enhancing CREB and BDNF expression. © 2015 John Wiley & Sons Ltd.

  8. Peripheral Nerve Regeneration and NGF-Dependent Neurite Outgrowth of Adult Sensory Neurons Converge on STAT3 Phosphorylation Downstream of Neuropoietic Cytokine Receptor gp130

    PubMed Central

    Quarta, Serena; Baeumer, Bastian E.; Scherbakov, Nadja; Andratsch, Manfred; Rose-John, Stefan; Dechant, Georg; Bandtlow, Christine E.

    2014-01-01

    After nerve injury, adult sensory neurons can regenerate peripheral axons and reconnect with their target tissue. Initiation of outgrowth, as well as elongation of neurites over long distances, depends on the signaling of receptors for neurotrophic growth factors. Here, we investigated the importance of gp130, the signaling subunit of neuropoietic cytokine receptors in peripheral nerve regeneration. After sciatic nerve crush, functional recovery in vivo was retarded in SNS-gp130−/− mice, which specifically lack gp130 in sensory neurons. Correspondingly, a significantly reduced number of free nerve endings was detected in glabrous skin from SNS-gp130−/− compared with control mice after nerve crush. Neurite outgrowth and STAT3 activation in vitro were severely reduced in cultures in gp130-deficient cultured neurons. Surprisingly, in neurons obtained from SNS-gp130−/− mice the increase in neurite length was reduced not only in response to neuropoietic cytokine ligands of gp130 but also to nerve growth factor (NGF), which does not bind to gp130-containing receptors. Neurite outgrowth in the absence of neurotrophic factors was partially rescued in gp130-deficient neurons by leptin, which activates STAT3 downstream of leptic receptor and independent of gp130. The neurite outgrowth response of gp130-deficient neurons to NGF was fully restored in the presence of leptin. Based on these findings, gp130 signaling via STAT3 activation is suggested not only to be an important regulator of peripheral nerve regeneration in vitro and in vivo, but as determining factor for the growth promoting action of NGF in adult sensory neurons. PMID:25253866

  9. Peripheral nerve regeneration and NGF-dependent neurite outgrowth of adult sensory neurons converge on STAT3 phosphorylation downstream of neuropoietic cytokine receptor gp130.

    PubMed

    Quarta, Serena; Baeumer, Bastian E; Scherbakov, Nadja; Andratsch, Manfred; Rose-John, Stefan; Dechant, Georg; Bandtlow, Christine E; Kress, Michaela

    2014-09-24

    After nerve injury, adult sensory neurons can regenerate peripheral axons and reconnect with their target tissue. Initiation of outgrowth, as well as elongation of neurites over long distances, depends on the signaling of receptors for neurotrophic growth factors. Here, we investigated the importance of gp130, the signaling subunit of neuropoietic cytokine receptors in peripheral nerve regeneration. After sciatic nerve crush, functional recovery in vivo was retarded in SNS-gp130(-/-) mice, which specifically lack gp130 in sensory neurons. Correspondingly, a significantly reduced number of free nerve endings was detected in glabrous skin from SNS-gp130(-/-) compared with control mice after nerve crush. Neurite outgrowth and STAT3 activation in vitro were severely reduced in cultures in gp130-deficient cultured neurons. Surprisingly, in neurons obtained from SNS-gp130(-/-) mice the increase in neurite length was reduced not only in response to neuropoietic cytokine ligands of gp130 but also to nerve growth factor (NGF), which does not bind to gp130-containing receptors. Neurite outgrowth in the absence of neurotrophic factors was partially rescued in gp130-deficient neurons by leptin, which activates STAT3 downstream of leptic receptor and independent of gp130. The neurite outgrowth response of gp130-deficient neurons to NGF was fully restored in the presence of leptin. Based on these findings, gp130 signaling via STAT3 activation is suggested not only to be an important regulator of peripheral nerve regeneration in vitro and in vivo, but as determining factor for the growth promoting action of NGF in adult sensory neurons. Copyright © 2014 the authors 0270-6474/14/3413222-12$15.00/0.

  10. A loss-of-function screen for phosphatases that regulate neurite outgrowth identifies PTPN12 as a negative regulator of TrkB tyrosine phosphorylation.

    PubMed

    Ambjørn, Malene; Dubreuil, Véronique; Miozzo, Federico; Nigon, Fabienne; Møller, Bente; Issazadeh-Navikas, Shohreh; Berg, Jacob; Lees, Michael; Sap, Jan

    2013-01-01

    Alterations in function of the neurotrophin BDNF are associated with neurodegeneration, cognitive decline, and psychiatric disorders. BDNF promotes axonal outgrowth and branching, regulates dendritic tree morphology and is important for axonal regeneration after injury, responses that largely result from activation of its tyrosine kinase receptor TrkB. Although intracellular neurotrophin (NT) signaling presumably reflects the combined action of kinases and phosphatases, little is known about the contributions of the latter to TrkB regulation. The issue is complicated by the fact that phosphatases belong to multiple independently evolved families, which are rarely studied together. We undertook a loss-of-function RNA-interference-based screen of virtually all known (254) human phosphatases to understand their function in BDNF/TrkB-mediated neurite outgrowth in differentiated SH-SY5Y cells. This approach identified phosphatases from diverse families, which either positively or negatively modulate BDNF-TrkB-mediated neurite outgrowth, and most of which have little or no previously established function related to NT signaling. "Classical" protein tyrosine phosphatases (PTPs) accounted for 13% of the candidate regulatory phosphatases. The top classical PTP identified as a negative regulator of BDNF-TrkB-mediated neurite outgrowth was PTPN12 (also called PTP-PEST). Validation and follow-up studies showed that endogenous PTPN12 antagonizes tyrosine phosphorylation of TrkB itself, and the downstream activation of ERK1/2. We also found PTPN12 to negatively regulate phosphorylation of p130cas and FAK, proteins with previously described functions related to cell motility and growth cone behavior. Our data provide the first comprehensive survey of phosphatase function in NT signaling and neurite outgrowth. They reveal the complexity of phosphatase control, with several evolutionarily unrelated phosphatase families cooperating to affect this biological response, and hence the

  11. A Loss-of-Function Screen for Phosphatases that Regulate Neurite Outgrowth Identifies PTPN12 as a Negative Regulator of TrkB Tyrosine Phosphorylation

    PubMed Central

    Ambjørn, Malene; Dubreuil, Véronique; Miozzo, Federico; Nigon, Fabienne; Møller, Bente; Issazadeh-Navikas, Shohreh; Berg, Jacob; Lees, Michael; Sap, Jan

    2013-01-01

    Alterations in function of the neurotrophin BDNF are associated with neurodegeneration, cognitive decline, and psychiatric disorders. BDNF promotes axonal outgrowth and branching, regulates dendritic tree morphology and is important for axonal regeneration after injury, responses that largely result from activation of its tyrosine kinase receptor TrkB. Although intracellular neurotrophin (NT) signaling presumably reflects the combined action of kinases and phosphatases, little is known about the contributions of the latter to TrkB regulation. The issue is complicated by the fact that phosphatases belong to multiple independently evolved families, which are rarely studied together. We undertook a loss-of-function RNA-interference-based screen of virtually all known (254) human phosphatases to understand their function in BDNF/TrkB-mediated neurite outgrowth in differentiated SH-SY5Y cells. This approach identified phosphatases from diverse families, which either positively or negatively modulate BDNF-TrkB-mediated neurite outgrowth, and most of which have little or no previously established function related to NT signaling. “Classical” protein tyrosine phosphatases (PTPs) accounted for 13% of the candidate regulatory phosphatases. The top classical PTP identified as a negative regulator of BDNF-TrkB-mediated neurite outgrowth was PTPN12 (also called PTP-PEST). Validation and follow-up studies showed that endogenous PTPN12 antagonizes tyrosine phosphorylation of TrkB itself, and the downstream activation of ERK1/2. We also found PTPN12 to negatively regulate phosphorylation of p130cas and FAK, proteins with previously described functions related to cell motility and growth cone behavior. Our data provide the first comprehensive survey of phosphatase function in NT signaling and neurite outgrowth. They reveal the complexity of phosphatase control, with several evolutionarily unrelated phosphatase families cooperating to affect this biological response, and hence

  12. 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

  13. A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin.

    PubMed

    Popova, Dina; Jacobsson, Stig O P

    2014-04-01

    The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10 days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity. In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Induction of Neurite Outgrowth in PC12 Cells Treated with Temperature-Controlled Repeated Thermal Stimulation

    PubMed Central

    Kudo, Tada-aki; Kanetaka, Hiroyasu; Mochizuki, Kentaro; Tominami, Kanako; Nunome, Shoko; Abe, Genji; Kosukegawa, Hiroyuki; Abe, Toshihiko; Mori, Hitoshi; Mori, Kazumi; Takagi, Toshiyuki; Izumi, Shin-ichi

    2015-01-01

    To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine. PMID:25879210

  15. Characteristic hexasaccharide sequences in octasaccharides derived from shark cartilage chondroitin sulfate D with a neurite outgrowth promoting activity.

    PubMed

    Nadanaka, S; Clement, A; Masayama, K; Faissner, A; Sugahara, K

    1998-02-06

    A mouse brain chondroitin sulfate (CS) proteoglycan, DSD-1-PG, bears the DSD-1 epitope and has neurite outgrowth promoting properties. Shark cartilage CS-C inhibits the interactions between the DSD-1-specific monoclonal antibody 473HD and the CS chains of the DSD-1-PG, which is expressed on the mouse glial cells (Faissner, A., Clement, A., Lochter, A., Streit, A., Mandl, C., and Schachner, M. (1994) J. Cell Biol. 126, 783-799). On the other hand, several hexasaccharides isolated from commercial shark cartilage CS-D, which contains a higher proportion of characteristic D units (GlcUA(2-sulfate)beta1-3GalNAc(6-sulfate)) as compared with CS-C, has the A-D tetrasaccharide sequence composed of an A disaccharide unit (GlcUAbeta1-3GalNAc(4-sulfate)) and a D disaccharide unit (Nadanaka, S. and Sugahara, K. (1997) Glycobiology 7, 253-263). In this study, the biological activities and the structure of shark cartilage CS-D were investigated. CS-D inhibited the interactions between monoclonal antibody 473HD and DSD-1-PG and also promoted neurite outgrowth of embryonic day 18 hippocampal neurons. Eight octasaccharide fractions were isolated from CS-D after partial digestion with bacterial chondroitinase ABC by means of gel filtration chromatography and anion-exchange high performance liquid chromotography to investigate the frequency and the arrangement of the A-D tetrasaccharide unit in the polymer sequence. Structural analysis performed by a combination of enzymatic digestions with 500-MHz 1H NMR spectroscopy demonstrated that the isolated octasaccharides shared the common core structure DeltaHexAalpha1-3GalNAcbeta1-4(GlcUAbeta1-3GalNAc)3 with four, five, and six sulfate esters at various hydroxyl groups in different combinations. In the structure, DeltaHexA and GlcUA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and glucuronic acid, respectively. No D-D tetrasaccharide sequence was found, and discrete D disaccharide units were demonstrated exclusively as A

  16. Conducting-polymer nanotubes improve electrical properties, mechanical adhesion, neural attachment, and neurite outgrowth of neural electrodes.

    PubMed

    Abidian, Mohammad Reza; Corey, Joseph M; Kipke, Daryl R; Martin, David C

    2010-02-05

    An in vitro comparison of conducting-polymer nanotubes of poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(pyrrole) (PPy) and to their film counterparts is reported. Impedance, charge-capacity density (CCD), tendency towards delamination, and neurite outgrowth are compared. For the same deposition charge density, PPy films and nanotubes grow relatively faster vertically, while PEDOT films and nanotubes grow more laterally. For the same deposition charge density (1.44 C cm(-2)), PPy nanotubes and PEDOT nanotubes have lower impedance (19.5 +/- 2.1 kOmega for PPy nanotubes and 2.5 +/- 1.4 kOmega for PEDOT nanotubes at 1 kHz) and higher CCD (184 +/- 5.3 mC cm(-2) for PPy nanotubes and 392 +/- 6.2 mC cm(-2) for PEDOT nanotubes) compared to their film counterparts. However, PEDOT nanotubes decrease the impedance of neural-electrode sites by about two orders of magnitude (bare iridium 468.8 +/- 13.3 kOmega at 1 kHz) and increase capacity of charge density by about three orders of magnitude (bare iridium 0.1 +/- 0.5 mC cm(-2)). During cyclic voltammetry measurements, both PPy and PEDOT nanotubes remain adherent on the surface of the silicon dioxide while PPy and PEDOT films delaminate. In experiments of primary neurons with conducting-polymer nanotubes, cultured dorsal root ganglion explants remain more intact and exhibit longer neurites (1400 +/- 95 microm for PPy nanotubes and 2100 +/- 150 microm for PEDOT nanotubes) than their film counterparts. These findings suggest that conducting-polymer nanotubes may improve the long-term function of neural microelectrodes.

  17. Conducting-Polymer Nanotubes Improve Electrical Properties, Mechanical Adhesion, Neural Attachment, and Neurite Outgrowth of Neural Electrodes

    PubMed Central

    Abidian, Mohammad Reza; Corey, Joseph M.; Kipke, Daryl R.; Martin, David C.

    2011-01-01

    An in vitro comparison of conducting-polymer nanotubes of poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(pyrrole) (PPy) and to their film counterparts is reported. Impedance, charge-capacity density (CCD), tendency towards delamination, and neurite outgrowth are compared. For the same deposition charge density, PPy films and nanotubes grow relatively faster vertically, while PEDOT films and nanotubes grow more laterally. For the same deposition charge density (1.44 C cm–2), PPy nanotubes and PEDOT nanotubes have lower impedance (19.5 ± 2.1 kΩ for PPy nanotubes and 2.5 ± 1.4 kΩ for PEDOT nanotubes at 1 kHz) and higher CCD (184 ± 5.3 mC cm–2 for PPy nanotubes and 392 ± 6.2 mC cm–2 for PEDOT nanotubes) compared to their film counterparts. However, PEDOT nanotubes decrease the impedance of neural-electrode sites by about two orders of magnitude (bare iridium 468.8 ± 13.3 kΩ at 1 kHz) and increase capacity of charge density by about three orders of magnitude (bare iridium 0.1 ± 0.5 mC cm–2). During cyclic voltammetry measurements, both PPy and PEDOT nanotubes remain adherent on the surface of the silicon dioxide while PPy and PEDOT films delaminate. In experiments of primary neurons with conducting-polymer nanotubes, cultured dorsal root ganglion explants remain more intact and exhibit longer neurites (1400 ± 95 μm for PPy nanotubes and 2100 ± 150 μm for PEDOT nanotubes) than their film counterparts. These findings suggest that conducting-polymer nanotubes may improve the long-term function of neural microelectrodes. PMID:20077424

  18. Effects of GnRH on Neurite Outgrowth, Neurofilament and Spinophilin Proteins Expression in Cultured Spinal Cord Neurons of Rat Embryos.

    PubMed

    Quintanar, J Luis; Calderón-Vallejo, Denisse; Hernández-Jasso, Irma

    2016-10-01

    It has been previously described the presence of GnRH receptor in spinal cord neurons of rat embryos and adult rats. However, the functional role of these receptors has not been studied. In this work, the effect of GnRH on neurite outgrowth and cytoskeletal protein expression in cultured spinal cord neurons of rat embryos was analyzed. Specifically, neurofilaments of 68 and 200 kDa by immunoblot assays and spinophilin mRNA expression by RT-PCR. Results show that GnRH stimulates neurite outgrowth in addition to an increase in neurofilaments and spinophilin expression. These findings suggest that GnRH may play a role as neuromodulator in neuronal plasticity and that could be considered as a potential factor for neuronal regeneration in spinal cord injuries.

  19. 1,25-dihydroxyvitamin D3 induces nerve growth factor, promotes neurite outgrowth and inhibits mitosis in embryonic rat hippocampal neurons.

    PubMed

    Brown, Jillanne; Bianco, John I; McGrath, John J; Eyles, Darryl W

    2003-06-05

    There is an accumulation of evidence implicating a role for vitamin D(3) in the developing brain. The receptor for this seco-steroid is expressed in both neurons and glial cells, it induces nerve growth factor (NGF) and it is a potent inhibitor of mitosis and promoter of differentiation in numerous cells. We have therefore assessed the direct effect of vitamin D(3) on mitosis, neurite outgrowth, as well as NGF production as a possible mediator of those effects, in developing neurons. Using cultured embryonic hippocampal cells and explants we found the addition of vitamin D(3) significantly decreases the percentage of cultured hippocampal cells undergoing mitosis in conjunction with increases in both neurite outgrowth and NGF production. The role of vitamin D(3) during brain development warrants closer scrutiny.

  20. Neurite outgrowth in human induced pluripotent stem cell-derived neurons as a high-throughput screen for developmental neurotoxicity or neurotoxicity.

    PubMed

    Ryan, Kristen R; Sirenko, Oksana; Parham, Fred; Hsieh, Jui-Hua; Cromwell, Evan F; Tice, Raymond R; Behl, Mamta

    2016-03-01

    Due to the increasing prevalence of neurological disorders and the large number of untested compounds in the environment, there is a need to develop reliable and efficient screening tools to identify environmental chemicals that could potentially affect neurological development. Herein, we report on a library of 80 compounds screened for their ability to inhibit neurite outgrowth, a process by which compounds may elicit developmental neurotoxicity, in a high-throughput, high-content assay using human neurons derived from induced pluripotent stem cells (iPSC). The library contains a diverse set of compounds including those that have been known to be associated with developmental neurotoxicity (DNT) and/or neurotoxicity (NT), environmental compounds with unknown neurotoxic potential (e.g., polycyclic aromatic hydrocarbons (PAHs) and flame retardants (FRs)), as well as compounds with no documented neurotoxic potential. Neurons were treated for 72h across a 6-point concentration range (∼0.3-100μM) in 384-well plates. Effects on neurite outgrowth were assessed by quantifying total outgrowth, branches, and processes. We also assessed the number ofviable cells per well. Concentration-response profiles were evaluated using a Hill model to derive benchmark concentration (BMC) values. Assay performance was evaluated using positive and negative controls and test replicates. Compounds were ranked by activity and selectivity (i.e., specific effects on neurite outgrowth in the absence of concomitant cytotoxicity) and repeat studies were conducted to confirm selectivity. Among the 80 compounds tested, 38 compounds were active, of which 16 selectively inhibited neurite outgrowth. Of these 16 compounds, 12 were known to cause DNT/NT and the remaining 4 compounds included 3 PAHs and 1 FR. In independent repeat studies, 14/16 selective compounds were reproducibly active in the assay, of which only 6 were selective for inhibition of neurite outgrowth. These 6 compounds were

  1. 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.

  2. Extremely Low-Frequency Electromagnetic Fields Promote In Vitro Neuronal Differentiation and Neurite Outgrowth of Embryonic Neural Stem Cells via Up-Regulating TRPC1

    PubMed Central

    Ma, Qinlong; Chen, Chunhai; Deng, Ping; Zhu, Gang; Lin, Min; Zhang, Lei; Xu, Shangcheng; He, Mindi; Lu, Yonghui; Duan, Weixia; Pi, Huifeng; Cao, Zhengwang; Pei, Liping; Li, Min; Liu, Chuan; Zhang, Yanwen; Zhong, Min; Zhou, Zhou; Yu, Zhengping

    2016-01-01

    Exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) can enhance hippocampal neurogenesis in adult mice. However, little is focused on the effects of ELF-EMFs on embryonic neurogenesis. Here, we studied the potential effects of ELF-EMFs on embryonic neural stem cells (eNSCs). We exposed eNSCs to ELF-EMF (50 Hz, 1 mT) for 1, 2, and 3 days with 4 hours per day. We found that eNSC proliferation and maintenance were significantly enhanced after ELF-EMF exposure in proliferation medium. ELF-EMF exposure increased the ratio of differentiated neurons and promoted the neurite outgrowth of eNSC-derived neurons without influencing astrocyes differentiation and the cell apoptosis. In addition, the expression of the proneural genes, NeuroD and Ngn1, which are crucial for neuronal differentiation and neurite outgrowth, was increased after ELF-EMF exposure. Moreover, the expression of transient receptor potential canonical 1 (TRPC1) was significantly up-regulated accompanied by increased the peak amplitude of intracellular calcium level induced by ELF-EMF. Furthermore, silencing TRPC1 expression eliminated the up-regulation of the proneural genes and the promotion of neuronal differentiation and neurite outgrowth induced by ELF-EMF. These results suggest that ELF-EMF exposure promotes the neuronal differentiation and neurite outgrowth of eNSCs via up-regulation the expression of TRPC1 and proneural genes (NeuroD and Ngn1). These findings also provide new insights in understanding the effects of ELF-EMF exposure on embryonic brain development. PMID:26950212

  3. Expression of glycogenes in differentiating human NT2N neurons. Downregulation of fucosyltransferase 9 leads to decreased Lewis(x) levels and impaired neurite outgrowth.

    PubMed

    Gouveia, Ricardo; Schaffer, Lana; Papp, Suzanne; Grammel, Nicolas; Kandzia, Sebastian; Head, Steven R; Kleene, Ralf; Schachner, Melitta; Conradt, Harald S; Costa, Júlia

    2012-12-01

    Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth. The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA. One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewis(x) motif (FUT9), polysialic acid (ST8SIA2 and ST8SIA4) and HNK-1 (B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewis(x) structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewis(x), as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression. These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewis(x) motif plays a functional role in neurite outgrowth from human neurons. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. 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.

  5. The Coffin-Lowry syndrome-associated protein RSK2 regulates neurite outgrowth through phosphorylation of phospholipase D1 (PLD1) and synthesis of phosphatidic acid.

    PubMed

    Ammar, Mohamed-Raafet; Humeau, Yann; Hanauer, André; Nieswandt, Bernard; Bader, Marie-France; Vitale, Nicolas

    2013-12-11

    More than 80 human X-linked genes have been associated with mental retardation and deficits in learning and memory. However, most of the identified mutations induce limited morphological alterations in brain organization and the molecular bases underlying neuronal clinical features remain elusive. We show here that neurons cultured from mice lacking ribosomal S6 kinase 2 (Rsk2), a model for the Coffin-Lowry syndrome (CLS), exhibit a significant delay in growth in a similar way to that shown by neurons cultured from phospholipase D1 (Pld1) knock-out mice. We found that gene silencing of Pld1 or Rsk2 as well as acute pharmacological inhibition of PLD1 or RSK2 in PC12 cells strongly impaired neuronal growth factor (NGF)-induced neurite outgrowth. Expression of a phosphomimetic PLD1 mutant rescued the inhibition of neurite outgrowth in PC12 cells silenced for RSK2, revealing that PLD1 is a major target for RSK2 in neurite formation. NGF-triggered RSK2-dependent phosphorylation of PLD1 led to its activation and the synthesis of phosphatidic acid at sites of neurite growth. Additionally, total internal reflection fluorescence microscopy experiments revealed that RSK2 and PLD1 positively control fusion of tetanus neurotoxin insensitive vesicle-associated membrane protein (TiVAMP)/VAMP-7 vesicles at sites of neurite outgrowth. We propose that the loss of function mutations in RSK2 that leads to CLS and neuronal deficits are related to defects in neuronal growth due to impaired RSK2-dependent PLD1 activity resulting in a reduced vesicle fusion rate and membrane supply.

  6. Epac and the high affinity rolipram binding conformer of PDE4 modulate neurite outgrowth and myelination using an in vitro spinal cord injury model

    PubMed Central

    Boomkamp, S D; McGrath, M A; Houslay, M D; Barnett, S C

    2014-01-01

    Background and Purpose cAMP and pharmacological inhibition of PDE4, which degrades it, are promising therapeutic targets for the treatment of spinal cord injury (SCI). Using our previously described in vitro SCI model, we studied the mechanisms by which cAMP modulators promote neurite outgrowth and myelination using enantiomers of the PDE4-specific inhibitor rolipram and other modulators of downstream signalling effectors. Experimental Approach Rat mixed neural cell myelinating cultures were cut with a scalpel and treated with enantiomers of the PDE4-specific inhibitor rolipram, Epac agonists and PKA antagonists. Neurite outgrowth, density and myelination were assessed by immunocytochemistry and cytokine levels analysed by qPCR. Key Results Inhibition of the high-affinity rolipram-binding state (HARBS), rather than the low-affinity rolipram binding state (LARBS) PDE4 conformer promoted neurite outgrowth and myelination. These effects were mediated through the activation of Epac and not through PKA. Expression of the chemokine CXCL10, known to inhibit myelination, was markedly elevated in astrocytes after Rho inhibition and this was blocked by inhibition of Rho kinase or PDE4. Conclusions and Implications PDE4 inhibitors targeted at the HARBS conformer or Epac agonists may provide promising novel targets for the treatment of SCI. Our study demonstrates the differential mechanisms of action of these compounds, as well as the benefit of a combined pharmacological approach and highlighting potential promising targets for the treatment of SCI. These findings need to be confirmed in vivo. PMID:24467222

  7. Moderate level exposure to magnetic nanodots encased in tunable poly(ethylene glycol) analouge biopolymer shell do not deleteriously affect neurite outgrowth.

    PubMed

    GhoshMitra, Somesree; Diercks, David R; Mills, Nathaniel C; Hynds, Di Anna L; Ghosh, Santaneel

    2013-12-01

    Recently, huge interest has been generated in investigating the possible therapeutic use of tunable magnetic nanostructures to overcome the existing challenges to treat central nervous system damage related conditions. However, several issues (e.g., biocompatibility or remote controlled actuation for multi-modal therapeutics) limit the use of conventional magnetic nanoparticles for biomedical applications. To address many of these shortcomings, we have synthesized a monodisperse nanoscale system consisting highly water dispersible magnetic nanodots encased in a remotely tunable polyethylene glycol analouge biopolymer shell. The monodisperse nature of the nanospheres, their response to external magnetic field and volumetric transition near physiological temperatures are very attractive, especially for drug delivery systems where triggered release is necessary. To further analyze the potential for combinatorial therapeutics for central nervous system damage related conditions, we have explored the efficiency of the uptake of nanospheres into pheochromocytoma cell line 12 (PC12) cells and assessed several additional measures of neurite outgrowth. We find that nanospheres were readily incorporated into the cytosolic compartment within 3 hours and did not alter the morphology of cellular processes compared to cells not exposed to nanospheres. Quantification of neurite outgrowth did not reveal any significant differences in neurite initiation or elongation between cells treated with moderate level nanomagnet exposure compared to control cultures under similar conditions. Thus, this study reports an attractive nano-scale system with great potential to deliver therapeutics to precise locations within the nervous system for axonal outgrowth and guidance.

  8. Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth.

    PubMed

    Miyamoto, Yuki; Yamauchi, Junji; Sanbe, Atsushi; Tanoue, Akito

    2007-02-15

    Small GTPases of the Rho family, Rho, Rac, and Cdc42, are critical regulators of the changes in the actin cytoskeleton. Rho GTPases are typically activated by Dbl-homology (DH)-domain-containing guanine nucleotide exchange factors (GEFs). Recent genetic and biochemical studies revealed a new type of GEF for the Rho GTPases. This family is composed of 11 genes, designated as Dock1 to Dock11, and is structurally divided into four classes Dock-A, -B, -C, and -D. Dock-A and -B subfamilies are typically GEFs specific for Rac1, while the Dock-D subfamily is specific for Cdc42. Here we show that Dock6, a member of the Dock-C subfamily, exchanges GDP for GTP for Rac1 and Cdc42 in vitro and in vivo. Furthermore, we find that, in mouse N1E-115 neuroblastoma cells, expression of Dock6 is increased following differentiation. Transfection of the catalytic Dock Homology Region-2 (DHR-2) domain of Dock6 promotes neurite outgrowth mediated by Rac1 and Cdc42. Conversely, knockdown of endogenous Dock6 by small interference RNA reduces activation of Rac1 and Cdc42 and neurite outgrowth. Taken together, these results suggest that Dock6 differs from all of the identified Dock180-related proteins, in that it is the GEF specific for both Rac1 and Cdc42 and may be one of physiological regulators of neurite outgrowth.

  9. Effects of sub-lethal neurite outgrowth inhibitory concentrations of chlorpyrifos oxon on cytoskeletal proteins and acetylcholinesterase in differentiating N2a cells.

    PubMed

    Flaskos, J; Nikolaidis, E; Harris, W; Sachana, M; Hargreaves, A J

    2011-11-01

    Previous work in our laboratory has shown that sub-lethal concentrations (1-10 μM) 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 μM) 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.

  10. Synapsin knockdown is associated with decreased neurite outgrowth, functional synaptogenesis impairment, and fast high-frequency neurotransmitter release.

    PubMed

    Brenes, Oscar; Giachello, Carlo Natale Giuseppe; Corradi, Anna Margherita; Ghirardi, Mirella; Montarolo, Pier Giorgio

    2015-10-01

    Synapsins (Syns) are an evolutionarily conserved family of synaptic vesicle-associated proteins related to fine tuning of synaptic transmission. Studies with mammals have partially clarified the different roles of Syns; however, the presence of different genes and isoforms and the development of compensatory mechanisms hinder accurate data interpretation. Here, we use a simple in vitro monosynaptic Helix neuron connection, reproducing an in vivo physiological connection as a reliable experimental model to investigate the effects of Syn knockdown. Cells overexpressing an antisense construct against Helix Syn showed a time-dependent decrease of Syn immunostaining, confirming protein loss. At the morphological level, Syn-silenced cells showed a reduction in neurite linear outgrowth and branching and in the size and number of synaptic varicosities. Functionally, Syn-silenced cells presented a reduced ability to form synaptic connections; however, functional chemical synapses showed similar basal excitatory postsynaptic potentials and similar short-term plasticity paradigms. In addition, Syn-silenced cells presented faster neurotransmitter release and decreased postsynaptic response toward the end of long tetanic presynaptic stimulations, probably related to an impairment of the synaptic vesicle trafficking resulting from a different vesicle handling, with an increased readily releasable pool and a compromised reserve pool.

  11. 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

  12. Disassembly of microtubules and inhibition of neurite outgrowth, neuroblastoma cell proliferation, and MAP kinase tyrosine dephosphorylation by dibenzyl trisulphide.

    PubMed

    Rösner, H; Williams, L A; Jung, A; Kraus, W

    2001-08-22

    Dibenzyl trisulphide (DTS), a main lipophilic compound in Petiveria alliacea L. (Phytolaccaceae), was identified as one of the active immunomodulatory compounds in extracts of the plant. To learn more about its biological activities and molecular mechanisms, we conducted one-dimensional NMR interaction studies with bovine serum albumin (BSA) and tested DTS and related compounds in two well-established neuronal cell-and-tissue culture systems. We found that DTS preferentially binds to an aromatic region of BSA which is rich in tyrosyl residues. In SH-SY5Y neuroblastoma cells, DTS attenuates the dephosphorylation of tyrosyl residues of MAP kinase (erk1/erk2). In the same neuroblastoma cell line and in Wistar 38 human lung fibroblasts, DTS causes a reversible disassembly of microtubules, but it did not affect actin dynamics. Probably due to the disruption of the microtubule dynamics, DTS also inhibits neuroblastoma cell proliferation and neurite outgrowth from spinal cord explants. Related dibenzyl compounds with none, one, or two sulphur atoms were found to be significantly less effective. These data confirmed that the natural compound DTS has a diverse spectrum of biological properties, including cytostatic and neurotoxic actions in addition to immunomodulatory activities.

  13. 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.

  14. The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of Rho and ROCK inhibitors on neurite outgrowth and myelination.

    PubMed

    Boomkamp, Stephanie D; Riehle, Mathis O; Wood, Jenifer; Olson, Michael F; Barnett, Susan C

    2012-03-01

    It is currently thought that treatment for spinal cord injury (SCI) will involve a combined pharmacological and biological approach; however, testing their efficacy in animal models of SCI is time-consuming and requires large animal cohorts. For this reason we have modified our myelinating cultures as an in vitro model of SCI and studied its potential as a prescreen for combined therapeutics. This culture comprises dissociated rat embryonic spinal cord cells plated onto a monolayer of astrocytes, which form myelinated axons interspaced with nodes of Ranvier. After cutting the culture, an initial cell-free area appears persistently devoid of neurites, accompanied over time by many features of SCI, including demyelination and reduced neurite density adjacent to the lesion, and infiltration of microglia and reactive astrocytes into the lesioned area. We tested a range of concentrations of the Rho inhibitor C3 transferase (C3) and ROCK inhibitor Y27632 that have been shown to promote SCI repair in vivo. C3 promoted neurite extension into the lesion and enhanced neurite density in surrounding areas but failed to induce remyelination. In contrast, while Y27632 did not induce significant neurite outgrowth, myelination adjacent to the lesion was dramatically enhanced. The effects of the inhibitors were concentration-dependent. Combined treatment with C3 and Y27632 had additive affects with an enhancement of neurite outgrowth and increased myelination adjacent to the lesion, demonstrating neither conflicting nor synergistic effects when coadministered. Overall, these results demonstrate that this culture serves as a useful tool to study combined strategies that promote CNS repair.

  15. Acetylcholine induces neurite outgrowth and modulates matrix metalloproteinase 2 and 9.

    PubMed

    Anelli, Tonino; Mannello, Ferdinando; Salani, Monica; Tonti, Gaetana A; Poiana, Giancarlo; Biagioni, Stefano

    2007-10-19

    The matrix metalloproteinases (MMPs), responsible for the degradation of extracellular matrix (ECM) proteins, may regulate brain cellular functions. Choline acetyltransferase (ChAT) transfected murine neuroblastoma cell line N18TG2, that synthesize acetylcholine and show enhancement of several neurospecific markers (i.e., sinapsin I, voltage gated Na(+) channels, high affinity choline uptake) and fiber outgrowth, were studied for the MMP regulation during neuronal differentiation. Zymography of N18TG2 culture medium revealed no gelatinolytic activity, whereas after carbachol treatment of cells both MMP-9 and activated MMP-2 forms were detected. ChAT-transfected clone culture medium contains three MMP forms at 230, 92, and 66kDa. Carbachol treatment increased MMP-2 and MMP-9 gene expression in N18TG2 cells and higher levels for both genes were also observed in ChAT transfected cells. The data are consistent with the hypothesis that acetylcholine brings about the activation of an autocrine loop modulating MMP expression.

  16. Nerve growth factor (NGF) and pro-NGF increase low-density lipoprotein (LDL) receptors in neuronal cells partly by different mechanisms: role of LDL in neurite outgrowth.

    PubMed

    Do, Hai Thi; Bruelle, Céline; Pham, Dan Duc; Jauhiainen, Matti; Eriksson, Ove; Korhonen, Laura T; Lindholm, Dan

    2016-01-01

    Low-density lipoprotein receptors (LDLRs) mediate the uptake of lipoprotein particles into cells, as studied mainly in peripheral tissues. Here, we show that nerve growth factor (NGF) increases LDLR levels in PC6.3 cells and in cultured septal neurons from embryonic rat brain. Study of the mechanisms showed that NGF enhanced transcription of the LDLR gene, acting mainly via Tropomyosin receptor kinase A receptors. Simvastatin, a cholesterol-lowering drug, also increased the LDLR expression in PC6.3 cells. In addition, pro-NGF and pro-brain-derived neurotrophic factor, acting via the p75 neurotrophin receptor (p75NTR) also increased LDLRs. We further observed that Myosin Regulatory Light Chain-Interacting Protein/Inducible Degrader of the LDLR (Mylip/Idol) was down-regulated by pro-NGF, whereas the other LDLR regulator, proprotein convertase subtilisin kexin 9 (PCSK9) was not significantly changed. On the functional side, NGF and pro-NGF increased lipoprotein uptake by neuronal cells as shown using diacetyl-labeled LDL. The addition of serum-derived lipoprotein particles in conjunction with NGF or simvastatin enhanced neurite outgrowth. Collectively, these results show that NGF and simvastatin are able to stimulate lipoprotein uptake by neurons with a positive effect on neurite outgrowth. Increases in LDLRs and lipoprotein particles in neurons could play a functional role during brain development, in neuroregeneration and after brain injuries. Nerve growth factor (NGF) and pro-NGF induce the expression of low-density lipoprotein receptors (LDLRs) in neuronal cells leading to increased LDLR levels. Pro-NGF also down-regulated myosin regulatory light chain-interacting protein/inducible degrader of the LDLR (Mylip/Idol) that is involved in the degradation of LDLRs. NGF acts mainly via Tropomyosin receptor kinase A (TrkA) receptors, whereas pro-NGF stimulates p75 neurotrophin receptor (p75NTR). Elevated LDLRs upon NGF and pro-NGF treatments enhanced lipoprotein uptake

  17. 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

  18. Neurite outgrowth stimulatory effects of myco synthesized AuNPs from Hericium erinaceus (Bull.: Fr.) Pers. on pheochromocytoma (PC-12) cells.

    PubMed

    Raman, Jegadeesh; Lakshmanan, Hariprasath; John, Priscilla A; Zhijian, Chan; Periasamy, Vengadesh; David, Pamela; Naidu, Murali; Sabaratnam, Vikineswary

    2015-01-01

    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. 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. 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. 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 stimulated neurite outgrowth. The water

  19. 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

  20. Lithium ion inhibits nerve growth factor-induced neurite outgrowth and phosphorylation of nerve growth factor-modulated microtubule-associated proteins

    PubMed Central

    1985-01-01

    LiCl (2.5-20 mM) reversibly suppressed nerve growth factor (NGF)- induced neurite outgrowth by cultured rat PC 12 pheochromocytoma cells. Similar concentrations of LiCl also reversibly blocked NGF-dependent regeneration of neurites by PC12 cells that had been primed by long- term pre-exposure to NGF and by cultured newborn mouse sympathetic neurons. In contrast, transcription-dependent responses of PC12 cells to NGF such as priming and induction of the NGF-inducible large external glycoprotein, occurred despite the presence of Li+. SDS PAGE analysis of total cellular phosphoproteins (labeled by 2-h exposure to 32P-orthophosphate) from neurite-bearing primed PC12 cells revealed that Li+ reversibly inhibited the phosphorylation of a band of Mr 64,000 that was barely detectable in NGF-untreated PC12 cells. However, Li+ did not appear to affect the labeling of other phosphoproteins in either NGF-primed or untreated PC12 cultures, nor did it affect the rapid increase in phosphorylation of several proteins that occurs when NGF is first added to unprimed cultures. Several criteria indicated that the NGF-inducible phosphoprotein of Mr 64,000 is a microtubule- associated protein (MAP). Of the NGF-inducible phosphorylated MAPs that have been detected in PC12 cells (Mr 64,000, 72,000, 80,000, and 320,000), several (Mr 64,000, 72,000, and 80,000) were found to be substantially less phosphorylated in the presence of Li+. Neither a phorbol ester tumor promotor nor permeant cAMP analogs reversed the inhibitory effects of Li+ on neurite outgrowth or on phosphorylation of the component of Mr 64,000. Microtubules are a major and required constituent of neurites, and MAPs may regulate the assembly and stability of neuritic microtubules. The observation that Li+ selectively inhibits NGF-induced neurite outgrowth and MAP phosphorylation suggests a possible causal relationship between these two events. PMID:4030895

  1. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3

    PubMed Central

    2013-01-01

    Background Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. Methods The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Results Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Conclusion Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health. PMID:24119256

  2. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3.

    PubMed

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

    2013-10-11

    Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health.

  3. Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells.

    PubMed

    Algarni, Alanood S; Hargreaves, Alan J; Dickenson, John M

    2017-03-15

    The PAC1 receptor and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 activity by the PAC1 receptor in retinoic acid-induced differentiating N2a neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. The role of TG2 in PAC1 receptor-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27). PACAP-27 mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON and R283 and by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), protein kinase C (Ro 31-8220), MEK1/2 (PD 98059), and removal of extracellular Ca(2+). Fluorescence microscopy demonstrated PACAP-27 induced in situ TG2 activity. TG2 inhibition blocked PACAP-27 induced attenuation of hypoxia-induced cell death and outgrowth of axon-like processes. TG2 activation and cytoprotection were also observed in human SH-SY5Y cells. Together, these results demonstrate that TG2 activity was stimulated downstream of the PAC1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC1 receptor.

  4. 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

  5. A genetic screen for neurite outgrowth mutants in Caenorhabditis elegans reveals a new function for the F-box ubiquitin ligase component LIN-23.

    PubMed Central

    Mehta, Nehal; Loria, Paula M; Hobert, Oliver

    2004-01-01

    Axon pathfinding and target recognition are highly dynamic and tightly regulated cellular processes. One of the mechanisms involved in regulating protein activity levels during axonal and synaptic development is protein ubiquitination. We describe here the isolation of several Caenorhabditis elegans mutants, termed eno (ectopic/erratic neurite outgrowth) mutants, that display defects in axon outgrowth of specific neuron classes. One retrieved mutant is characterized by abnormal termination of axon outgrowth in a subset of several distinct neuron classes, including ventral nerve cord motor neurons, head motor neurons, and mechanosensory neurons. This mutant is allelic to lin-23, which codes for an F-box-containing component of an SCF E3 ubiquitin ligase complex that was previously shown to negatively regulate postembryonic cell divisions. We demonstrate that LIN-23 is a broadly expressed cytoplasmically localized protein that is required autonomously in neurons to affect axon outgrowth. Our newly isolated allele of lin-23, a point mutation in the C-terminal tail of the protein, displays axonal outgrowth defects similar to those observed in null alleles of this gene, but does not display defects in cell cycle regulation. We have thus defined separable activities of LIN-23 in two distinct processes, cell cycle control and axon patterning. We propose that LIN-23 targets distinct substrates for ubiquitination within each process. PMID:15082545

  6. Epac1 interacts with importin β1 and controls neurite outgrowth independently of cAMP and Rap1

    PubMed Central

    Baameur, Faiza; Singhmar, Pooja; Zhou, Yong; Hancock, John F.; Cheng, Xiaodong; Heijnen, Cobi J.; Kavelaars, Annemieke

    2016-01-01

    Exchange protein directly activated by cAMP-1 (Epac1) is a cAMP sensor that regulates multiple cellular functions including cellular migration, proliferation and differentiation. Classically, Epac1 is thought to exert its effects through binding of cAMP leading to a conformational change in Epac1 and its accumulation at the plasma membrane (PM) where it activates Rap1. In search for regulators of Epac1 activity, we show here that importin β1 (impβ1) is an Epac1 binding partner that prevents PM accumulation of Epac1. We demonstrate that in the absence of impβ1, endogenous as well as overexpressed Epac1 accumulate at the PM. Moreover, agonist-induced PM translocation of Epac1 leads to dissociation of Epac1 from impβ1. Localization of Epac1 at the PM in the absence of impβ1, requires residue R82 in its DEP domain. Notably, the PM accumulation of Epac1 in the absence of impβ1 does not require binding of cAMP to Epac1 and does not result in Rap1 activation. Functionally, PM accumulation of Epac1, an Epac1 mutant deficient in cAMP binding, or an Epac1 mutant tethered to the PM, is sufficient to inhibit neurite outgrowth. In conclusion, we uncover a cAMP-independent function of Epac1 at the PM and demonstrate that impβ1 controls subcellular localization of Epac1. PMID:27808165

  7. Synergistic induction of neurite outgrowth by nerve growth factor or epidermal growth factor and interleukin-6 in PC12 cells.

    PubMed

    Wu, Y Y; Bradshaw, R A

    1996-05-31

    Native PC12 cells respond differentially to nerve growth factor (NGF) but not interleukin-6 (IL-6); PC12-E2 cells, a stable variant, respond to both stimuli (and more rapidly to NGF). Neither responds to epidermal growth factor (EGF). NGF primarily induces the RAS/extracellular signal-regulated kinase (ERK) pathway and IL-6 activates a JAK (Janus tyrosine kinase)/STAT (signal transducers and activators of transcription) response. EGF also stimulates RAS/ERK but in a transient manner. When either cell type is treated with combinations of NGF, EGF, and IL-6, at concentrations that produce modest or no response, a substantial augmentation of neurite outgrowth is observed. With PC12-E2 cells, a subthreshold concentration of IL-6 increases NGF response by approximately 2-3-fold after 1-2 days; the increase with EGF is more pronounced. Native PC12 cells show even greater synergistic effects with NGF and IL-6. The most dramatic effect was observed with low levels of EGF, where IL-6 increased the percentage of responsive cells from zero to approximately 60% after 3 days. In addition, two neural-specific transcripts, GAP-43 and SCG-10, are synergistically increased by the combinations of growth factors. Importantly, IL-6 does not enhance ERK phosphorylation in the presence of either NGF or EGF. In contrast, NGF and EGF, in the presence or absence of IL-6, cause mobility shifts of Stat3 that are consistent with serine phosphorylations. Although these modifications do not lead to activation and translocation by themselves, in the presence of the tyrosine phosphorylation induced by IL-6, they may play a role in the synergistic responses. These observations suggest a differentially regulated two-stage mechanism for the differentiative response of PC12 cells to NGF.

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

    PubMed Central

    Zhang, Kai; Duan, Liting; Ong, Qunxiang; Lin, Ziliang; Varman, Pooja Mahendra; Sung, Kijung; Cui, Bianxiao

    2014-01-01

    It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network. PMID:24667437

  9. Up-regulation of miR-26a promotes neurite outgrowth and ameliorates apoptosis by inhibiting PTEN in bupivacaine injured mouse dorsal root ganglia.

    PubMed

    Cui, Changlei; Xu, Gong; Qiu, Jinpeng; Fan, Xiushuang

    2015-08-01

    Local anesthetic of bupivacaine may inhibit neurite outgrowth and induce apoptosis in mouse dorsal root ganglia (DRG) neurons. In this work, we intended to investigate the functional role of microRNA 26a (miR-26a) in regulating bupivacaine-induced nerve injury in DRG neurons. DRG neurons were extracted from C57BL/6 mice and cultured in vitro. Bupivacaine was applied in vitro and it induced apoptosis, inhibited neurite growth, and significantly down-regulated miR-26a gene in DRG neurons. MiR-26a mimic was then used to up-regulate miR-26a expression in DRG neurons. We found that miR-26a up-regulation promoted neurite outgrowth and reduced apoptosis in bupivacaine-injured DRG neurons. Luciferase assay and Western blot confirmed that Phosphatase and tensin homolog (PTEN) was down-stream target of miR-26a in DRG neurons. Ectopic PTEN up-regulation was then able to reverse the protective effect of miR-26a overexpression on bupivacaine-induced nerve injury in DRG neurons. Overall, this work demonstrated that miR-26a had a functional role in regulating bupivacaine-induced nerve injury in DRG neurons. Up-regulating miR-26a to suppress PTEN signaling pathway may be an effective method to protect local anesthetic-induced nerve injury in spinal cord.

  10. Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells*

    PubMed Central

    Etoh, Kan; Fukuda, Mitsunori

    2015-01-01

    The small GTPase Rab35 is a molecular switch for membrane trafficking that regulates a variety of cellular events. We previously showed that Rab35 promotes neurite outgrowth of nerve growth factor-stimulated PC12 cells through interaction with centaurin-β2 (also called ACAP2). Centaurin-β2 is the only Rab35-binding protein reported thus far that exclusively recognizes Rab35 and does not recognize any of the other 59 Rabs identified in mammals, but the molecular basis for the exclusive specificity of centaurin-β2 for Rab35 has remained completely unknown. In this study, we performed deletion and mutation analyses and succeeded in identifying the residues of Rab35 and centaurin-β2 that are crucial for formation of a Rab35·centaurin-β2 complex. We found that two threonine residues (Thr-76 and Thr-81) in the switch II region of Rab35 are responsible for binding centaurin-β2 and that the same residues are dispensable for Rab35 recognition by other Rab35-binding proteins. We also determined the minimal Rab35-binding site of centaurin-β2 and identified two asparagine residues (Asn-610 and Asn-691) in the Rab35-binding site as key residues for its specific Rab35 recognition. We further showed by knockdown-rescue approaches that neither a centaurin-β2 binding-deficient Rab35(T76S/T81A) mutant nor a Rab35 binding-deficient centaurin-β2(N610A/N691A) mutant supported neurite outgrowth of PC12 cells, thereby demonstrating the functional significance of the Rab35/centaurin-β2 interaction during neurite outgrowth of PC12 cells. PMID:25694427

  11. 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.

  12. ERK5 Activity Is Required for Nerve Growth Factor-induced Neurite Outgrowth and Stabilization of Tyrosine Hydroxylase in PC12 Cells*

    PubMed Central

    Obara, Yutaro; Yamauchi, Arata; Takehara, Shin; Nemoto, Wataru; Takahashi, Maho; Stork, Philip J. S.; Nakahata, Norimichi

    2009-01-01

    Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt2cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 μm, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt2cAMP, but BIX02188 and BIX02189, at 30 μm, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt2cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt2cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process. PMID:19581298

  13. Inhibition of mTOR by Rapamycin Results in Auditory Hair Cell Damage and Decreased Spiral Ganglion Neuron Outgrowth and Neurite Formation In Vitro

    PubMed Central

    Leitmeyer, Katharina; Glutz, Andrea; Radojevic, Vesna; Setz, Cristian; Huerzeler, Nathan; Bumann, Helen; Bodmer, Daniel; Brand, Yves

    2015-01-01

    Rapamycin is an antifungal agent with immunosuppressive properties. Rapamycin inhibits the mammalian target of rapamycin (mTOR) by blocking the mTOR complex 1 (mTORC1). mTOR is an atypical serine/threonine protein kinase, which controls cell growth, cell proliferation, and cell metabolism. However, less is known about the mTOR pathway in the inner ear. First, we evaluated whether or not the two mTOR complexes (mTORC1 and mTORC2, resp.) are present in the mammalian cochlea. Next, tissue explants of 5-day-old rats were treated with increasing concentrations of rapamycin to explore the effects of rapamycin on auditory hair cells and spiral ganglion neurons. Auditory hair cell survival, spiral ganglion neuron number, length of neurites, and neuronal survival were analyzed in vitro. Our data indicates that both mTOR complexes are expressed in the mammalian cochlea. We observed that inhibition of mTOR by rapamycin results in a dose dependent damage of auditory hair cells. Moreover, spiral ganglion neurite number and length of neurites were significantly decreased in all concentrations used compared to control in a dose dependent manner. Our data indicate that the mTOR may play a role in the survival of hair cells and modulates spiral ganglion neuronal outgrowth and neurite formation. PMID:25918725

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

    PubMed

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

    2014-11-15

    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.

  15. Ephrin-A4 inhibits sensory neurite outgrowth and is regulated by neonatal skin wounding.

    PubMed

    Moss, Andrew; Alvares, Debie; Meredith-Middleton, Jacqueta; Robinson, Michelle; Slater, Rebeccah; Hunt, Stephen P; Fitzgerald, Maria

    2005-11-01

    The mechanisms for directing and organising sensory axons within developing skin remain largely unknown. The present study provides the first evidence that signalling occurs between A-ephrins and Eph-A receptors during the development of rat cutaneous sensory innervation both during normal development and following skin injury. Specifically, our data indicate that ephrin-A4 mRNA and protein are expressed in the epidermis during late embryogenesis and the early postnatal period (E16-P3), and expression is significantly down-regulated postnatally. In addition, Eph-A receptors are expressed on dorsal root ganglia (DRG) cells at birth. The pattern of ephrin-A4 expression is mirrored by epidermal innervation, so that sensory terminals are restricted to epidermal regions devoid of ephrin-A4 but increase as ephrin-A4 expression subsides postnatally. Neonatal skin wounding causes sensory hyperinnervation and a differential screen of wounded vs. nonwounded skin revealed down-regulation of epidermal ephrin-A4 following neonatal skin wounding. Expression studies showed that this down-regulation is below the wound and coincides exactly with the onset of hyperinnervation. In vitro experiments show a function for ephrin-A4-Fc in inhibiting rat DRG neuronal growth and guidance when presented as either substratum-bound stripes of ephrin-A4-Fc or as soluble clustered proteins. In conclusion, these observations suggest that the Eph family ligand ephrin-A4 has an inhibitory influence on neonatal cutaneous nerve terminals from DRG sensory neurons in the hindlimb, and may serve to prevent inappropriate innervation of cutaneous regions. In addition, the absence of ephrin-A4 following neonatal skin wounding may play a critical permissive role in the sprouting response.

  16. The role of CD4-dependent signaling in interleukin-16 induced c-Fos expression and facilitation of neurite outgrowth in cerebellar granule neurons.

    PubMed

    Fenster, Catherine P; Chisnell, Hope K; Fry, Carl R; Fenster, Steven D

    2010-11-26

    Neuronal interleukin 16 (NIL-16) is the larger neural-specific splice variant of the interleukin-16 (IL16) gene and shows restricted expression to post-mitotic neurons of the mammalian hippocampus and cerebellum. Although the N-terminus of NIL-16 is unique to the neuronal variant, the C-terminus is identical to pro-IL-16, the IL-16 precursor expressed primarily in T-cells. IL-16 was originally described as a proinflammatory cytokine and has diverse immunoregulatory effects which involve signaling through CD4. NIL-16-expressing neurons can secrete IL-16 and may express CD4; moreover, treatment of cultured cerebellar granule neurons (CGCs) with IL-16 increases the expression of c-Fos, an immediate-early gene which transcriptionally regulates genes directing survival, proliferation, and growth. Taken together, we hypothesize that IL-16 functions as a neuroregulatory cytokine which signals through neuronal CD4 receptors. In this study, we investigated the role of CD4 in IL-16-induced c-Fos expression in CGCs, as well as the effects of IL-16 on neuronal survival and growth. We detected components involved in IL-16-signaling in lymphocytes, including CD4 and the associated tyrosine kinase p56(lck), in CGCs using qRT-PCR and immunoblotting. We also show that IL-16 induces c-Fos expression in wild-type CGCs, but not CD4-deficient CGCs or following inhibition of p56(lck). Finally, treatment of CGCs with IL-16 enhanced neurite outgrowth, an effect also observed in CD4-deficient CGCs. Taken together, our results indicate that IL-16-signaling affects neuronal gene expression and growth through CD4-dependent and independent pathways. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  17. RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells

    PubMed Central

    Tam, See-Ying; Lilla, Jennifer N.; Chen, Ching-Cheng; Kalesnikoff, Janet; Tsai, Mindy

    2015-01-01

    Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells. PMID:26588713

  18. RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells.

    PubMed

    Tam, See-Ying; Lilla, Jennifer N; Chen, Ching-Cheng; Kalesnikoff, Janet; Tsai, Mindy

    2015-01-01

    Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.

  19. GABAergic Control of Neurite Outgrowth and Remodeling During Development and Adult Neurogenesis: General Rules and Differences in Diverse Systems

    PubMed Central

    Sernagor, Evelyne; Chabrol, François; Bony, Guillaume; Cancedda, Laura

    2010-01-01

    During development, Gamma-aminobutyric acidergic (GABAergic) neurons mature at early stages, long before excitatory neurons. Conversely, GABA reuptake transporters become operative later than glutamate transporters. GABA is therefore not removed efficiently from the extracellular domain and it can exert significant paracrine effects. Hence, GABA-mediated activity is a prominent source of overall neural activity in developing CNS networks, while neurons extend dendrites and axons, and establish synaptic connections. One of the unique features of GABAergic functional plasticity is that in early development, activation of GABAA receptors results in depolarizing (mainly excitatory) responses and Ca2+ influx. Although there is strong evidence from several areas of the CNS that GABA plays a significant role in neurite growth not only during development but also during adult neurogenesis, surprisingly little effort has been made into putting all these observations into a common framework in an attempt to understand the general rules that regulate these basic and evolutionary well-conserved processes. In this review, we discuss the current knowledge in this important field. In order to decipher common, universal features and highlight differences between systems throughout development, we compare findings about dendritic proliferation and remodeling in different areas of the nervous system and species, and we also review recent evidence for a role in axonal elongation. In addition to early developmental aspects, we also consider the GABAergic role in dendritic growth during adult neurogenesis, extending our discussion to the roles played by GABA during dendritic proliferation in early developing networks versus adult, well established networks. PMID:20428495

  20. Regulation of NGF-driven neurite outgrowth by Ins(1,4,5)P3 kinase is specifically associated with the two isoenzymes Itpka and Itpkb in a model of PC12 cells.

    PubMed

    Koenig, Sandra; Moreau, Colette; Dupont, Geneviève; Scoumanne, Ariane; Erneux, Christophe

    2015-07-01

    Four inositol phosphate kinases catalyze phosphorylation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 ] to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4 ]: these enzymes comprise three isoenzymes of inositol 1,4,5-trisphosphate 3-kinase (Itpk), referred to as Itpka, Itpkb and Itpkc, and the inositol polyphosphate multikinase (IPMK). The four enzymes that act on Ins(1,4,5)P3 are all expressed in rat pheochromocytoma PC12 cells, a model that is used to study neurite outgrowth induced by nerve growth factor (NGF). We compared the effect of over-expression of the four GFP-tagged kinases on NGF-induced neurite outgrowth. Our data show that over-expression of the Itpka and Itpkb isoforms inhibits NGF-induced neurite outgrowth, but over-expression of Itpkc and IPMK does not. Surprisingly, over-expression of the N-terminal F-actin binding domain of Itpka, which lacks catalytic activity, was as effective at inhibiting neurite outgrowth as the full-length enzyme. Neurite length was also significantly decreased in cells over-expressing Itpka and Itpkb but not Itpkc or IPMK. This result did not depend on the over-expression level of any of the kinases. PC12 cells over-expressing GFP-tagged kinase-dead mutants Itpka/b have shorter neurites than GFP control cells. The decrease in neurite length was never as pronounced as observed with wild-type GFP-tagged Itpka/b. Finally, the percentage of neurite-bearing cells was increased in cells over-expressing the membranous type I Ins(1,4,5)P3 5-phosphatase. We conclude that Itpka and Itpkb inhibit neurite outgrowth through both F-actin binding and localized Ins(1,4,5)P3 3-kinase activity. Itpkc and IPMK do not influence neurite outgrowth or neurite length in this model.

  1. Lion's Mane, Hericium erinaceus and Tiger Milk, Lignosus rhinocerotis (Higher Basidiomycetes) Medicinal Mushrooms Stimulate Neurite Outgrowth in Dissociated Cells of Brain, Spinal Cord, and Retina: An In Vitro Study.

    PubMed

    Samberkar, Snehlata; Gandhi, Sivasangkary; Naidu, Murali; Wong, Kah-Hui; Raman, Jegadeesh; Sabaratnam, Vikineswary

    2015-01-01

    Neurodegenerative disease is defined as a deterioration of the nervous system in the intellectual and cognitive capabilities. Statistics show that more than 80-90 million individuals age 65 and above in 2050 may be affected by neurodegenerative conditions like Alzheimer's and Parkinson's disease. Studies have shown that out of 2000 different types of edible and/or medicinal mushrooms, only a few countable mushrooms have been selected until now for neurohealth activity. Hericium erinaceus is one of the well-established medicinal mushrooms for neuronal health. It has been documented for its regenerative capability in peripheral nerve. Another mushroom used as traditional medicine is Lignosus rhinocerotis, which has been used for various illnesses. It has been documented for its neurite outgrowth potential in PC12 cells. Based on the regenerative capabilities of both the mushrooms, priority was given to select them for our study. The aim of this study was to investigate the potential of H. erinaceus and L. rhinocerotis to stimulate neurite outgrowth in dissociated cells of brain, spinal cord, and retina from chick embryo when compared to brain derived neurotrophic factor (BDNF). Neurite outgrowth activity was confirmed by the immu-nofluorescence method in all tissue samples. Treatment with different concentrations of extracts resulted in neuronal differentiation and neuronal elongation. H. erinaceus extract at 50 µg/mL triggered neurite outgrowth at 20.47%, 22.47%, and 21.70% in brain, spinal cord, and retinal cells. L. rhinocerotis sclerotium extract at 50 µg/mL induced maximum neurite outgrowth of 20.77% and 24.73% in brain and spinal cord, whereas 20.77% of neurite outgrowth was observed in retinal cells at 25 µg/mL, respectively.

  2. Multi-porous electroactive poly(L-lactic acid)/polypyrrole composite micro/nano fibrous scaffolds promote neurite outgrowth in PC12 cells.

    PubMed

    Yu, Qiaozhen; Xu, Shuiling; Zhang, Kuihua; Shan, Yongming

    2013-01-05

    In this study, poly(L-lactic acid)/ammonium persulfate doped-polypyrrole composite fibrous scaffolds with moderate conductivity were produced by combining electrospinning with in situ polymerization. PC12 cells were cultured on these fibrous scaffolds and their growth following electrical stimulation (0-20.0 μA stimulus intensity, for 1-4 days) was observed using inverted light microscopy, and scanning electron microscopy coupled with the MTT cell viability test. The results demonstrated that the poly(L-lactic acid)/ammonium persulfate doped-polypyrrole fibrous scaffold was a dual multi-porous micro/nano fibrous scaffold. An electrical stimulation with a current intensity 5.0-10.0 μA for about 2 days enhanced neuronal growth and neurite outgrowth, while a high current intensity (over 15.0 μA) suppressed them. These results indicate that electrical stimulation with a moderate current intensity for an optimum time frame can promote neuronal growth and neurite outgrowth in an intensity- and time-dependent manner.

  3. Effects of Extremely Low Frequency Magnetic Field on Neurite Outgrowth of PC12 and PC12D Cells and Evaluation by Image Analysis

    NASA Astrophysics Data System (ADS)

    Sakanishi, Akio; Takatsuki, Hideyo; Yoshikoshi, Akio; Fujiwara, Yasuyoshi

    2004-05-01

    A pheochromocytoma cell (PC12), and its derivative (PC12D), differentiate to nervelike cells in culture with the nerve growth factor (NGF) and forskolin respectively. We introduced a morphological factor σ=L/2(π A)1/2 for quantitating neurite outgrowth under a microscope in the presence of extremely low-frequency (ELF) magnetic fields for 22 hours, where L and A are the contour length and the area of the cells in clump determined using an image-analysis system. ELF magnetic fields B1 were generated with a single coil or double coils in Helmholtz configuration together with static fields B0 of -53, -20 and 67 μT. σ increased with increasing NGF or forskolin level at B0=-53 μT (geomagnetism), in agreement with the cytometric observation of micrographs. With the addition of an AC field B1 at 60 Hz (100 μT > B1 > 3 μT rms) to B0, neurite outgrowth represented by σ was depressed for PC12 and stimulated for PC12D. We discuss the cyclotron resonance and the ion parametric resonance models.

  4. MicroRNA-127 targeting of mitoNEET inhibits neurite outgrowth, induces cell apoptosis and contributes to physiological dysfunction after spinal cord transection

    PubMed Central

    He, Qin-Qin; Xiong, Liu-Lin; Liu, Fei; He, Xiang; Feng, Guo-Ying; Shang, Fei-Fei; Xia, Qing-Jie; Wang, You-Cui; Qiu, De-Lu; Luo, Chao-Zhi; Liu, Jia; Wang, Ting-Hua

    2016-01-01

    Neuroregeneration and apoptosis are two important pathophysiologic changes after spinal cord injury (SCI), but their underlying mechanisms remain unclear. MicroRNAs (miRNAs) play a crucial role in the regulation of neuroregeneration and neuronal apoptosis, research areas that have been greatly expanded in recent years. Here, using miRNA arrays to profile miRNA transcriptomes, we demonstrated that miR-127-3p was significantly down-regulated after spinal cord transection (SCT). Then, bioinformatics analyses and experimental detection showed that miR-127-3p exhibited specific effects on the regulation of neurite outgrowth and the induction of neuronal apoptosis by regulating the expression of the mitochondrial membrane protein mitoNEET. Moreover, knockdown of MitoNEET leaded to neuronal loss and apoptosis in primary cultured spinal neurons. This study therefore revealed that miR-127-3p, which targets mitoNEET, plays a vital role in regulating neurite outgrowth and neuronal apoptosis after SCT. Thus, modificatioin of the mitoNEET expression, such as mitoNEET activition may provide a new strategy for the treatment of SCI in preclinical trials. PMID:27748416

  5. 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.

  6. Multi-porous electroactive poly(L-lactic acid)/polypyrrole composite micro/nano fibrous scaffolds promote neurite outgrowth in PC12 cells☆

    PubMed Central

    Yu, Qiaozhen; Xu, Shuiling; Zhang, Kuihua; Shan, Yongming

    2013-01-01

    In this study, poly(L-lactic acid)/ammonium persulfate doped-polypyrrole composite fibrous scaffolds with moderate conductivity were produced by combining electrospinning with in situ polymerization. PC12 cells were cultured on these fibrous scaffolds and their growth following electrical stimulation (0–20.0 μA stimulus intensity, for 1–4 days) was observed using inverted light microscopy, and scanning electron microscopy coupled with the MTT cell viability test. The results demonstrated that the poly(L-lactic acid)/ammonium persulfate doped-polypyrrole fibrous scaffold was a dual multi-porous micro/nano fibrous scaffold. An electrical stimulation with a current intensity 5.0–10.0 μA for about 2 days enhanced neuronal growth and neurite outgrowth, while a high current intensity (over 15.0 μA) suppressed them. These results indicate that electrical stimulation with a moderate current intensity for an optimum time frame can promote neuronal growth and neurite outgrowth in an intensity- and time-dependent manner. PMID:25206369

  7. The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells.

    PubMed

    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(Cip1), a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G(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.

  8. 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.

  9. Ferulic Acid Protects Against Lead Acetate-Induced Inhibition of Neurite Outgrowth by Upregulating HO-1 in PC12 Cells: Involvement of ERK1/2-Nrf2 Pathway.

    PubMed

    Yu, Chun-Lei; Zhao, Xue-Mei; Niu, Ying-Cai

    2016-11-01

    Prenatal lead exposure is associated with poor intellectual development in children. However, there are few breakthroughs in therapeutic intervention of developmental lead neurotoxicity. The aim of this study is to evaluate the hypothesis that ferulic acid-mediated promotion of neurite outgrowth following lead exposure might mainly result from its antioxidant capability by extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Exposure of PC12 cells to lead acetate inhibits neurite outgrowth and causes oxidative stress as measured by ROS, LPO, GSH/GSSG, and NAD(+)/NADH. FA treatment significantly, although not completely, protected the cells against lead acetate-induced neurite outgrowth inhibition. The effects of FA could be blocked by PD98059, zinc protoporphyrin (Zn-PP), and Nrf2 shRNA. In addition, FA induced heme oxygenase 1 (HO-1) gene expression, enhanced antioxidant response element (ARE) promoter activity, promoted ERK1/2 phosphorylation, and Nrf2 translocation in PC12 cells exposed to lead acetate. ERK1/2 locate upstream of Nrf2 and regulate Nrf2-dependent HO-1 expression in antioxidative effects of FA. Our results suggest that FA is a promising candidate for treatment of developmental lead neurotoxicity. These promising findings warrant future investigation evaluating the FA-mediated potentiation of neurite outgrowth following lead exposure in vivo.

  10. Reaching Out to Send a Message: Proteins Associated with Neurite Outgrowth and Neurotransmission are Altered with Age in the Long-Lived Naked Mole-Rat.

    PubMed

    Triplett, Judy C; Swomley, Aaron M; Kirk, Jessime; Grimes, Kelly M; Lewis, Kaitilyn N; Orr, Miranda E; Rodriguez, Karl A; Cai, Jian; Klein, Jon B; Buffenstein, Rochelle; Butterfield, D Allan

    2016-07-01

    Aging is the greatest risk factor for developing neurodegenerative diseases, which are associated with diminished neurotransmission as well as neuronal structure and function. However, several traits seemingly evolved to avert or delay age-related deterioration in the brain of the longest-lived rodent, the naked mole-rat (NMR). The NMR remarkably also exhibits negligible senescence, maintaining an extended healthspan for ~75 % of its life span. Using a proteomic approach, statistically significant changes with age in expression and/or phosphorylation levels of proteins associated with neurite outgrowth and neurotransmission were identified in the brain of the NMR and include: cofilin-1; collapsin response mediator protein 2; actin depolymerizing factor; spectrin alpha chain; septin-7; syntaxin-binding protein 1; synapsin-2 isoform IIB; and dynamin 1. We hypothesize that such changes may contribute to the extended lifespan and healthspan of the NMR.

  11. Mitogen-activated protein kinases regulate expression of neuronal nitric oxide synthase and neurite outgrowth via non-classical retinoic acid receptor signaling in human neuroblastoma SH-SY5Y cells.

    PubMed

    Fujibayashi, Tatsuya; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Shudo, Koichi; Katsuki, Hiroshi

    2015-10-01

    We have previously shown that retinoic acid receptor (RAR) stimulation by an agonist Am80 recruits nitric oxide-dependent signaling via increased expression of neuronal nitric oxide synthase (nNOS) in rat midbrain slice cultures. Using neuroblastoma SH-SY5Y cells, here we investigated the mechanisms of RAR-induced nNOS expression, together with relationship between nNOS expression and neurite outgrowth. Am80 promoted neurite outgrowth, which was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K; LY294002), c-Jun N-terminal kinase (JNK; SP600125) and p38 mitogen-activated protein kinase (p38 MAPK; SB203580). A selective nNOS inhibitor 3-bromo-nitroindazole also suppressed Am80-induced neurite outgrowth. Am80-induced increase in nNOS protein expression was attenuated by LY294002, SP600125 and SB203580, whereas increase in nNOS mRNA expression was attenuated only by LY294002. Am80-induced activation of JNK and p38 MAPK was blocked by LY294002, suggesting that these kinases acted downstream of PI3K. We also confirmed that DAX1, a nuclear receptor reported to regulate nNOS expression, was up-regulated in response to Am80. siRNA-mediated knockdown of DAX1 abrogated Am80-induced nNOS expression and neurite outgrowth. These results reveal for the first time that nNOS expression is crucial for RAR-mediated neurite outgrowth, and that non-genomic signaling such as JNK and p38 MAPK is involved in RAR-mediated nNOS expression.

  12. TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling.

    PubMed

    Heiser, Jeanine H; Schuwald, Anita M; Sillani, Giacomo; Ye, Lian; Müller, Walter E; Leuner, Kristina

    2013-11-01

    The non-selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. Alterations in synaptic plasticity are considered to play an important role in the pathogenesis of depression. Beside several other proteins, TRPC6 channels regulate synaptic plasticity. This study demonstrates that different pathways including Ras/MEK/ERK, PI3K/Akt, and CAMKIV are involved in the improvement of synaptic plasticity by the TRPC6 activator hyperforin, the antidepressant active constituent of St. John

  13. Enhancement of neurite outgrowth in PC12 cells stimulated with cyclic AMP and NGF by 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), novel lipophilic ascorbate derivatives.

    PubMed

    Zhou, Xiaohua; Tai, Akihiro; Yamamoto, Itaru

    2003-03-01

    It has been shown that ascorbate (AsA) and its stable derivative, ascorbic acid 2-O-alpha-glucoside (AA-2G), do not elicit neurite outgrowth in PC12 cells. However, these ascorbates are synergistically enhanced by both dibutyryl cyclic AMP (Bt(2)cAMP)- and nerve growth factor (NGF)-induced neurite outgrowth in this model. In the present study, the effects of a series of novel lipophilic ascorbate derivatives, 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), on neurite outgrowth induced by Bt(2)cAMP and NGF were examined in PC12 cells. We found that all the tested acylated ascorbate derivatives enhanced neurite formation induced by both agents in a dose-dependent manner. Of the 6-Acyl-AA-2G derivatives, 6-octanoyl ascorbic acid 2-O-alpha-glucoside (6-Octa-AA-2G) enhanced the Bt(2)cAMP-induced phosphorylated MAPK p44 and p42 expression. A alpha-glucosidase inhibitor, castanospermine, completely abrogated the promotion of neurite outgrowth and MAPK expression by 6-Octa-AA-2G. Addition of 6-Octa-AA-2G (0.5 mM) to PC12 cells caused a rapid and significant increase in intracellular AsA content, which reached a maximum and was maintained from 12 to 24 h after the culture. These findings suggest that 6-Acyl-AA-2G is rapidly hydrolyzed to AsA within the cell and enhances neurite differentiation through the interaction with the inducer-activated MAPK pathway.

  14. Neural cell adhesion molecule NrCAM is expressed in the mammalian inner ear and modulates spiral ganglion neurite outgrowth in an in vitro alternate choice assay.

    PubMed

    Brand, Yves; Sung, Michael; Pak, Kwang; Chavez, Eduardo; Wei, Eric; Radojevic, Vesna; Bodmer, Daniel; Ryan, Allen F

    2015-04-01

    Neuron-glial-related cell adhesion molecule (NrCAM) is a neuronal cell adhesion molecule involved in neuron-neuron and neuron-glial adhesion as well as directional signaling during axonal cone growth. NrCAM has been shown to be involved in several cellular processes in the central and peripheral nervous systems, including neurite outgrowth, axonal pathfinding and myelination, fasciculation of nerve fibers, and cell migration. This includes sensory systems such as the eye and olfactory system. However, there are no reports on the expression/function of NrCAM in the auditory system. The aim of the present study was to elucidate the occurrence of NrCAM in the mammalian cochlea and its role in innervation of the auditory end organ. Our work indicates that NrCAM is highly expressed in the developing mammalian cochlea (position consistent with innervation). Moreover, we found that NrCAM, presented in stripe micropatterns, provide directional cues to neonatal rat inner ear spiral ganglion neurites in vitro. Our results are consistent with a role for NrCAM in the pathfinding of spiral ganglion dendrites toward their hair cell targets in the sensory epithelium.

  15. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Angiotensin II type 2 receptor (AT2 R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons.

    PubMed

    Anand, U; Facer, P; Yiangou, Y; Sinisi, M; Fox, M; McCarthy, T; Bountra, C; Korchev, Y E; Anand, P

    2013-08-01

    The angiotensin II (AngII) receptor subtype 2 (AT2 R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. We used immunostaining with characterized antibodies to study the localization of AT2 R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2 R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. AT2 R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2 R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50  = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1 R antagonist losartan had no effect on capsaicin responses. AT2 R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2 R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. AT2 R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting. © 2012 European Federation of International Association for the Study of Pain Chapters.

  17. Angiotensin II type 2 receptor (AT2R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons

    PubMed Central

    Anand, U; Facer, P; Yiangou, Y; Sinisi, M; Fox, M; McCarthy, T; Bountra, C; Korchev, YE; Anand, P

    2013-01-01

    Background The angiotensin II (AngII) receptor subtype 2 (AT2R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. Methods We used immunostaining with characterized antibodies to study the localization of AT2R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. Results AT2R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1R antagonist losartan had no effect on capsaicin responses. AT2R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. Conclusions AT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting. PMID:23255326

  18. Fibrin functionalization with synthetic adhesive ligands interacting with α6β1 integrin receptor enhance neurite outgrowth of embryonic stem cell-derived neural stem/progenitors.

    PubMed

    Silva, Joana; Bento, Ana R; Barros, Daniela; Laundos, Tiago L; Sousa, Susana R; Quelhas, Pedro; Sousa, Mónica M; Pêgo, Ana P; Amaral, Isabel F

    2017-09-01

    To enhance fibrin hydrogel affinity towards pluripotent stem cell-derived neural stem/progenitor cells (NSPCs) and its capacity to support NSPC migration and neurite extension, we explored the tethering of synthetic peptides engaging integrin α6β1, a cell receptor enriched in NSPCs. Six α6β1 integrin ligands were tested for their ability to support integrin α6β1-mediated adhesion of embryonic stem cell-derived NSPCs (ES-NSPs) and sustain ES-NSPC viability, migration, and neuronal differentiation. Due to their better performance, peptides T1, HYD1, and A5G81 were immobilized into fibrin and functionalized gels characterized in terms of peptide binding efficiency, structure and viscoelastic properties. Tethering of T1 or HYD1 successfully enhanced cell outgrowth from ES-NSPC neurospheres (up to 2.4-fold increase), which exhibited a biphasic response to peptide concentration. Inhibition assays evidenced the involvement of α6β1 and α3β1 integrins in mediating radial outgrowth on T1-/HYD1-functionalized gels. Fibrin functionalization also promoted neurite extension of single ES-NSPCs in fibrin, without affecting cell proliferation and neuronal differentiation. Finally, HYD1-functionalized gels were found to provide a permissive environment for axonal regeneration, leading up to a 2.0-fold increase in neurite extension from rat dorsal root ganglia explants as compared to unmodified fibrin, and to significant improved locomotor function after spinal cord injury (complete transection), along with a trend toward a higher area positive for growth associated protein 43 (marker for axonal growth cone formation). Our results suggest that conjugation of α6β1 integrin-binding motifs is of interest to increase the biofunctionality of hydrogels used in 3D platforms for ES-NSPC culture and potentially, in matrix-assisted ES-NSPC transplantation. Impact statement: The transplantation of NSPCs derived from pluripotent stem cells holds much promise for the treatment of

  19. Diabetes impairs an interleukin-1β-dependent pathway that enhances neurite outgrowth through JAK/STAT3 modulation of mitochondrial bioenergetics in adult sensory neurons

    PubMed Central

    2013-01-01

    Background A luminex-based screen of cytokine expression in dorsal root ganglia (DRG) and nerve of type 1 diabetic rodents revealed interleukin-1 (IL-1α) and IL-1β to be significantly depressed. We, therefore, tested the hypothesis that impaired IL-1α and IL-1β expression in DRG may contribute to aberrant axon regeneration and plasticity seen in diabetic sensory neuropathy. In addition, we determined if these cytokines could optimize mitochondrial bioenergetics since mitochondrial dysfunction is a key etiological factor in diabetic neuropathy. Results Cytokines IL-1α and IL-1β were reduced 2-fold (p<0.05) in DRG and/or nerve of 2 and 5 month streptozotocin (STZ)-diabetic rats. IL-2 and IL-10 were unchanged. IL-1α and IL-1β induced similar 2 to 3-fold increases in neurite outgrowth in cultures derived from control or diabetic rats (p<0.05). STAT3 phosphorylation on Tyr705 or Ser727 was depressed in DRG from STZ-diabetic mice and treatment of cultures derived from STZ-diabetic rats with IL-1β for 30 min raised phosphorylation of STAT3 on Tyr705 and Ser727 by 1.5 to 2-fold (p<0.05). shRNA-based or AG490 inhibition of STAT3 activity or shRNA blockade of endogenous IL-1β expression completely blocked neurite outgrowth. Cultured neurons derived from STZ-diabetic mice were treated for 24 hr with IL-1β and maximal oxygen consumption rate and spare respiratory capacity, both key measures of bioenergetic fidelity that were depressed in diabetic compared with control neurons, were enhanced 2-fold. This effect was blocked by AG490. Conclusions Endogenous synthesis of IL-1β is diminished in nerve tissue in type 1 diabetes and we propose this defect triggers reduced STAT3 signaling and mitochondrial function leading to sup-optimal axonal regeneration and plasticity. PMID:24152426

  20. Novel degradable co-polymers of polypyrrole support cell proliferation and enhance neurite out-growth with electrical stimulation.

    PubMed

    Durgam, Hymavathi; Sapp, Shawn; Deister, Curt; Khaing, Zin; Chang, Emily; Luebben, Silvia; Schmidt, Christine E

    2010-01-01

    Synthetic polymers such as polypyrrole (PPy) are gaining significance in neural studies because of their conductive properties. We evaluated two novel biodegradable block co-polymers of PPy with poly(epsilon-caprolactone) (PCL) and poly(ethyl cyanoacrylate) (PECA) for nerve regeneration applications. PPy-PCL and PPy-PECA co-polymers can be processed from solvent-based colloidal dispersions and have essentially the same or greater conductivity (32 S/cm for PPy-PCL, 19 S/cm for PPy-PECA) compared to the PPy homo-polymer (22 S/cm). The PPy portions of the co-polymers permit electrical stimulation whereas the PCL or PECA blocks enable degradation by hydrolysis. For in vitro tests, films were prepared on polycarbonate sheets by air brushing layers of dispersions and pressing the films. We characterized the films for hydrolytic degradation, electrical conductivity, cell proliferation and neurite extension. The co-polymers were sufficient to carry out electrical stimulation of cells without the requirement of a metallic conductor underneath the co-polymer film. In vitro electrical stimulation of PPy-PCL significantly increased the number of PC12 cells bearing neurites compared to unstimulated PPy-PCL. For in vivo experiments, the PPy co-polymers were coated onto the inner walls of nerve guidance channels (NGCs) made of the commercially available non-conducting biodegradable polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV). The NGCs were implanted in a 10 mm defect made in the sciatic nerve of rats, and harvested after 8 weeks. Histological staining showed axonal growth. The studies indicated that these new conducting degradable biomaterials have good biocompatibility and support proliferation and growth of PC12 cells in vitro (with and without electrical stimulation) and neurons in vivo (without electrical stimulation).

  1. Nucleus-localized 21.5-kDa myelin basic protein promotes oligodendrocyte proliferation and enhances neurite outgrowth in coculture, unlike the plasma membrane-associated 18.5-kDa isoform.

    PubMed

    Smith, Graham S T; Samborska, Bożena; Hawley, Steven P; Klaiman, Jordan M; Gillis, Todd E; Jones, Nina; Boggs, Joan M; Harauz, George

    2013-03-01

    The classic myelin basic protein (MBP) family of central nervous system (CNS) myelin arises from transcription start site 3 of the Golli (gene of oligodendrocyte lineage) complex and comprises splice isoforms ranging in nominal molecular mass from 14 kDa to (full-length) 21.5 kDa. We have determined here a number of distinct functional differences between the major 18.5-kDa and minor 21.5-kDa isoforms of classic MBP with respect to oligodendrocyte (OLG) proliferation. We have found that, in contrast to 18.5-kDa MBP, 21.5-kDa MBP increases proliferation of early developmental immortalized N19-OLGs by elevating the levels of phosphorylated ERK1/2 and Akt1 kinases and of ribosomal protein S6. Coculture of N2a neuronal cells with N19-OLGs transfected with the 21.5-kDa isoform (or conditioned medium from), but not the 18.5-kDa isoform, caused the N2a cells to have increased neurite outgrowth and process branching complexity. These roles were dependent on subcellular localization of 21.5-kDa MBP to the nucleus and on the exon II-encoded segment, suggesting that the nuclear localization of early minor isoforms of MBP may play a crucial role in regulating and/or initiating myelin and neuronal development in the mammalian CNS.

  2. Brain-derived neurotrophic factor promotes vesicular glutamate transporter 3 expression and neurite outgrowth of dorsal root ganglion neurons through the activation of the transcription factors Etv4 and Etv5.

    PubMed

    Liu, Dong; Liu, Zhen; Liu, Huaxiang; Li, Hao; Pan, Xinliang; Li, Zhenzhong

    2016-03-01

    Brain-derived neurotrophic factor (BDNF) is critical for sensory neuron survival and is necessary for vesicular glutamate transporter 3 (VGLUT3) expression. Whether the transcription factors Etv4 and Etv5 are involved in these BDNF-induced effects remains unclear. In the present study, primary cultured dorsal root ganglion (DRG) neurons were used to test the link between BDNF and transcription factors Etv4 and Etv5 on VGLUT3 expression and neurite outgrowth. BDNF promoted the mRNA and protein expression of Etv4 and Etv5 in DRG neurons. These effects were blocked by extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor PD98059 but not phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or phospholipase C-γ (PLC-γ) inhibitor U73122. Etv4 siRNA and Etv5 siRNA effectively blocked the VGLUT3 expression and neurite elongation induced by BNDF. The overexpression of Etv4 or Etv5 potentiated the effects of BNDF-induced neurite elongation and growth-associated protein 43 (GAP-43), medium neurofilament (NF-M), and light neurofilament (NF-L) expression while these effects could be inhibited by Etv4 and Etv5 siRNA. These data imply that Etv4 and Etv5 are essential transcription factors in modulating BDNF/TrkB signaling-mediated VGLUT3 expression and neurite outgrowth. BDNF, through the ERK1/2 signaling pathway, activates Etv4 and Etv5 to initiate GAP-43 expression, promote neurofilament (NF) protein expression, induce neurite outgrowth, and mediate VGLUT3 expression for neuronal function improvement. The biological effects initiated by BDNF/TrkB signaling linked to E26 transformation-specific (ETS) transcription factors are important to elucidate neuronal differentiation, axonal regeneration, and repair in various pathological states.

  3. The interaction between cell adhesion molecule L1, matrix metalloproteinase 14, and adenine nucleotide translocator at the plasma membrane regulates L1-mediated neurite outgrowth of murine cerebellar neurons.

    PubMed

    Loers, Gabriele; Makhina, Tatjana; Bork, Ute; Dörner, Andrea; Schachner, Melitta; Kleene, Ralf

    2012-03-14

    We have identified the adenine nucleotide translocator (ANT) isoforms ANT1 and ANT2 that are present in the plasma membrane of mouse cerebellar neurons as novel binding partners of the cell adhesion molecule L1. The direct interaction between ANT and L1 is mediated by sites within the fibronectin type III domains of L1 and the first and third extracellular loops of the ANT proteins. We also show that L1 interacts with the ANT binding partner matrix metalloprotease 14 (MMP14) and that the ANT proteins bind directly to the L1 interaction partner glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, we provide evidence that the functional interplay between L1, ANT proteins, MMP14, and GAPDH at the plasma membrane mediates L1-induced neurite outgrowth of cerebellar neurons. Disruption of this interplay by ANT inhibitors, ANT-derived synthetic peptides, and/or function-blocking MMP14 and ANT antibodies leads to alterations in L1-dependent neurite outgrowth. Stimulation of L1-mediated signaling in cerebellar neurons triggers transient ATP secretion via ANT proteins and leads to transient src family-dependent tyrosine phosphorylation of L1, ANT1, ANT2, and MMP14. Thus, our results indicate that plasma membrane-localized ANT1 and ANT2 regulate L1-mediated neurite outgrowth in conjunction with MMP14.

  4. Laminins 2 (alpha2beta1gamma1, Lm-211) and 8 (alpha4beta1gamma1, Lm-411) are synthesized and secreted by tooth pulp fibroblasts and differentially promote neurite outgrowth from trigeminal ganglion sensory neurons.

    PubMed

    Fried, Kaj; Sime, Wondossen; Lillesaar, Christina; Virtanen, Ismo; Tryggvasson, Karl; Patarroyo, Manuel

    2005-07-15

    The tooth pulp innervation originates from the trigeminal ganglion (TG) and represents an illustrative example of tissue targeting by sensory nerves. Pulpal fibroblasts strongly promote neurite outgrowth from TG neurons in vitro. In the present study, we have investigated the possible participation of laminins (LNs), potent neuritogenic extracellular matrix components. Immunohistochemistry of human tooth pulp demonstrated expression of LN alpha1, alpha2, alpha4, alpha5, beta1 and gamma1, and laminin-binding integrin alpha3, alpha6, beta1 and beta4 chains in nerves. Though faintly stained for laminins in situ, pulpal fibroblasts reacted, once cultured and permeabilized, with antibodies to LN alpha2, alpha4, beta1 and gamma1 chains by flow cytometry. The cells also expressed the corresponding mRNAs and were able to assemble and secrete LN-2 (alpha2beta1gamma1, Lm-211) and LN-8 (alpha4beta1gamma1, Lm-411). LN-8 displayed a chondroitin sulphate (CS) modification in its alpha4 chain. In functional assays, mouse LN-1 (alpha1beta1gamma1, Lm-111) and recombinant human (rh) LN-8, but not native or rhLN-2, strongly promoted neurite outgrowth from TG neurons, mimicking the effect of cultured pulp fibroblast. Altogether, the results indicate that LN-2 and LN-8 are synthesized by tooth pulp fibroblasts and differentially promote neurite outgrowth from TG neurons. LN-8 may contribute to sensory innervation of teeth and other tissues during development and/or regeneration.

  5. Functionalized Collagen Scaffold Neutralizing the Myelin-Inhibitory Molecules Promoted Neurites Outgrowth in Vitro and Facilitated Spinal Cord Regeneration in Vivo.

    PubMed

    Li, Xing; Han, Jin; Zhao, Yannan; Ding, Wenyong; Wei, Jianshu; Han, Sufang; Shang, Xianping; Wang, Bin; Chen, Bing; Xiao, Zhifeng; Dai, Jianwu

    2015-07-01

    Research has demonstrated that many myelin-associated inhibitory molecules jointly contribute to the failure of adult spinal cord regeneration. Therapies comprehensively targeting the multiple inhibitory nature of the injured spinal cord are being concerned. Here, two collagen-binding proteins, CBD-EphA4LBD and CBD-PlexinB1LBD, were constructed, respectively, to neutralize the axon guidance molecules ephrinB3 and sema4D that inhibit the regeneration of nerve fibers. The two neutralizing proteins have proven their ability to specifically bind collagen and to continuously release from collagen scaffolds. They could also promote neurites outgrowth of cerebellar granular neurons and dorsal root ganglion neurons in vitro. Subsequently, the functionalized collagen scaffolds by physically absorbing NEP1-40 and immobilizing CBD-EphA4LBD and CBD-PlexinB1LBD were transplanted into a rat T10 complete spinal cord transection model. Our results showed that rats that received the treatment of transplanting the functionalized collagen scaffold exhibited great advantage on axonal regeneration and locomotion recovery after spinal cord injury.

  6. Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth.

    PubMed

    Pacary, Emilie; Tixier, Emmanuelle; Coulet, Florence; Roussel, Simon; Petit, Edwige; Bernaudin, Myriam

    2007-07-01

    This study demonstrates that the Rho-kinase (ROCK) inhibitor, Y-27632, potentiates not only the effect of cobalt chloride (CoCl(2)) but also that of deferoxamine, another HIF-1 inducer, on mesenchymal stem cell (MSC) neuronal differentiation. HIF-1 is essential for CoCl(2)+/-Y-27632-induced MSC neuronal differentiation, since agents inhibiting HIF-1 abolish the changes of morphology and cell cycle arrest-related gene or protein expressions (p21, cyclin D1) and the increase of neuronal marker expressions (Tuj1, NSE). Y-27632 potentiates the CoCl(2)-induced decrease of cyclin D1 and nestin expressions, the increase of HIF-1 activation and EPO expression, and decreases pVHL expression. Interestingly, CoCl(2) decreases RhoA expression, an effect potentiated by Y-27632, revealing crosstalk between HIF-1 and RhoA/ROCK pathways. Moreover, we demonstrate a synergistic effect of CoCl(2) and Y-27632 on neurosphere differentiation into neurons and PC12 neurite outgrowth underlining that a co-treatment targeting both HIF-1 and ROCK pathways might be relevant to differentiate stem cells into neurons.

  7. rAAV-mediated delivery of brain-derived neurotrophic factor promotes neurite outgrowth and protects neurodegeneration in focal ischemic model.

    PubMed

    Zhang, Jingyu; Yu, Zhigang; Yu, Zhiqiang; Yang, Zichao; Zhao, Hong; Liu, Luran; Zhao, Jiexu

    2011-06-20

    Stroke is one of the neurological diseases which lead to permanently neuronal damage after temporary or long-term occlusion of vessels or after heart attack. However, there are few efficient strategies to prevent or treat this kind of insult in clinical because the consequence is irreversible and could be long-lasting after the onset of stroke. Gene therapy especially using viral system has long been addressed to be of great potential to reduce the damage. Here, we generated recombinant adeno-associated virus (rAAV) carrying brain-derived neurotrophic factor (BDNF) gene. Cells infected with rAAV-BDNF could be able to produce functional BDNF which promoted neurite outgrowth and protected neurons from apoptosis induced by serum deprivation. Further more, single injection of rAAV showed neuroprotection against cell death in focal ischemic model. These results showed that rAAV-mediated gene delivery is functional, which shed light to the future application of viral system-based gene therapy in clinical.

  8. Time course study of Aβ formation and neurite outgrowth disruption in differentiated human neuroblastoma cells exposed to H2O2: protective role of autophagy.

    PubMed

    Ashabi, Ghorbangol; Ahmadiani, Abolhassan; Abdi, Azadeh; Abraki, Shahnaz Babaei; Khodagholi, Fariba

    2013-09-01

    Here, we tried to elucidate the possible role of autophagy against H2O2 and Amyloid beta (Aβ) induced neurotoxicity using retinoic acid differentiated SH-SY5Y cells. We found that H2O2 disrupted neurite outgrowth concomitant with production of Aβ. Furthermore, we showed that H2O2 could increase the apoptotic factors such as Bax/Bcl-2 ratio, caspase-3 level, and PARP activity in a time course manner. These findings were confirmed by acridine orange/ethidium bromide and Hoechst staining. In addition, we observed that H2O2 led to conversion of LC3 protein from LC3I to LC3II and an increase in autophagy flux. Autophagy factors including LC3B, Atg7, and Atg12 increased and reached their highest level after 2h of insulting and then dropped to a lower level. Our results showed that autophagy could internalize and degrade intra- and extracellular Aβ after 3h treatment with H2O2. However, the remaining amount of Aβ accelerated morphological atrophy and, as a result, increased neuronal death (apoptosis). Inhibition of autophagy influx, using 3-methyl-adenine, increased intra- and extracellular levels of Aβ, providing more proof for a protective role of autophagy against oxidative stress. Further studies can shed light on the important role of autophagy by finding new pathways involved in Aβ degeneration.

  9. Identification of a potent and selective σ₁ receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells.

    PubMed

    Rossi, Daniela; Pedrali, Alice; Urbano, Mariangela; Gaggeri, Raffaella; Serra, Massimo; Fernández, Leyden; Fernández, Michael; Caballero, Julio; Ronsisvalle, Simone; Prezzavento, Orazio; Schepmann, Dirk; Wuensch, Bernhard; Peviani, Marco; Curti, Daniela; Azzolina, Ornella; Collina, Simona

    2011-11-01

    Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (σ) ligands based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was studied. Novel arylalkenylamines generally possess high σ(1) receptor affinity (K(i) values <25 nM) and good σ(1)/σ(2) selectivity (K(i)σ(2) >100). Particularly, the piperidine derivative (E)-17 and its arylalkylamine analog (R,S)-33 were observed to be excellent σ(1) receptor ligands (K(i)=0.70 and 0.86 nM, respectively) and to display significantly high selectivity over σ(2), μ-, and κ-opioid receptors and phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptors. Moreover in PC12 cells (R,S)-33 promoted the nerve growth factor (NGF)-induced neurite outgrowth and elongation. Co-administration of the selective σ(1) receptor antagonist BD-1063 totally counteracted this effect, confirming that σ(1) receptors are involved in the (R,S)-33 modulation of the NGF effect in PC12 cells and suggesting a σ(1) agonist profile. As a part of our work, a threedimensional σ(1) pharmacophore model was also developed employing GALAHAD methodology. Only active compounds were used for deriving this model. The model included two hydrophobes and a positive nitrogen as relevant features and it was able to discriminate between molecules with and without affinity toward σ(1) receptor subtype.

  10. Vitamin B(12), a chlorophyll-related analog to pheophytin a from marine brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells.

    PubMed

    Ina, Atsutoshi; Kamei, Yuto

    2006-11-01

    We previously isolated an analog to chlorophyll-related compounds, pheophytin a, from the marine brown alga Sargassum fulvellum and demonstrated that it is a neurodifferentiation compound. In the current study, we investigated the effects of the pheophytin a analog vitamin B(12) on PC12 cell differentiation. In the presence of a low level of nerve growth factor (10 ng ml(-1)), vitamin B(12 )demonstrated neurite outgrowth-promoting activity in PC12 cells. The effect was dose-dependent in the range of 6-100 muM. In the absence of nerve growth factor, vitamin B(12) did not promote differentiation. To investigate the mechanism for this effect, we conducted differentiation assays and western blot analysis with signal transduction inhibitors and found that vitamin B(12) did not promote PC12 cell differentiation in the presence of K252a or U0126 inhibitors. These results suggest that vitamin B(12 )stimulates PC12 cell differentiation through enhancement of the mitogen-activated protein kinase signal transduction pathway, which is also induced by nerve growth factor. Thus, vitamin B(12) may be a good candidate for treatment of neurodegenerative diseases such as Alzheimer's disease.

  11. IMPACT is a developmentally regulated protein in neurons that opposes the eukaryotic initiation factor 2α kinase GCN2 in the modulation of neurite outgrowth.

    PubMed

    Roffé, Martín; Hajj, Glaucia N M; Azevedo, Hátylas F; Alves, Viviane S; Castilho, Beatriz A

    2013-04-12

    The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.

  12. Ect2, an ortholog of Drosophila's pebble, negatively regulates neurite outgrowth in neuroblastoma × glioma hybrid NG108-15 cells.

    PubMed

    Tsuji, Takahiro; Higashida, Chiharu; Yoshida, Yasumasa; Islam, Mohammad Saharul; Dohmoto, Mitsuko; Koizumi, Keita; Higashida, Haruhiro

    2011-07-01

    To identify genes required for brain development, we previously performed in vivo RNA interference (RNAi) screening in Drosophila embryos. We identified pebble as a gene that disrupts development of the Drosophila nervous system. Although pebble has been shown to be involved in neuronal development of Drosophila in several screens, the involvement of Ect2, a mammalian ortholog of pebble, in mammalian neuronal development has not been addressed. To examine the role of Ect2 in neuronal differentiation, we performed Ect2 RNAi in the mouse neuroblastoma × rat glioma NG108-15 cell line. Depletion of Ect2 resulted in an increased proportion of binucleate cells and morphological differentiation of NG108-15 cells characterized by the outgrowth of neurites. These morphological changes were correlated with an increased level of acetylcholine esterase mRNA. In addition, expression of Ect2 was decreased in differentiated NG108-15 cells induced by dibutyryl cyclic AMP. These findings indicate that Ect2 negatively regulates the differentiation of NG108-15 cells and suggest that Ect2 may play a role in neuronal differentiation and brain development in vivo.

  13. Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells.

    PubMed

    Borodinsky, Laura N; Coso, Omar A; Fiszman, Mónica L

    2002-03-01

    In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.

  14. Comparison of Neurite Outgrowth Induced by Erythropoietin (EPO) and Carbamylated Erythropoietin (CEPO) in Hippocampal Neural Progenitor Cells.

    PubMed

    Oh, Dong Hoon; Lee, In Young; Choi, Miyeon; Kim, Seok Hyeon; Son, Hyeon

    2012-08-01

    A previous animal study has shown the effects of erythropoietin (EPO) and its non-erythropoietic carbamylated derivative (CEPO) on neurogenesis in the dentate gyrus. In the present study, we sought to investigate the effect of EPO on adult hippocampal neurogenesis, and to compare the ability of EPO and CEPO promoting dendrite elongation in cultured hippocampal neural progenitor cells. Two-month-old male BALB/c mice were given daily injections of EPO (5 U/g) for seven days and were sacrificed 12 hours after the final injection. Proliferation assays demonstrated that EPO treatment increased the density of bromodeoxyuridine (BrdU)-labeled cells in the subgranular zone (SGZ) compared to that in vehicle-treated controls. Functional differentiation studies using dissociated hippocampal cultures revealed that EPO treatment also increased the number of double-labeled BrdU/microtubule-associated protein 2 (MAP2) neurons compared to those in vehicle-treated controls. Both EPO and CEPO treatment significantly increased the length of neurites and spine density in MAP2(+) cells. In summary, these results provide evidences that EPO and CEPO promote adult hippocampal neurogenesis and neuronal differentiation. These suggest that EPO and CEPO could be a good candidate for treating neuropsychiatric disorders such as depression and anxiety associated with neuronal atrophy and reduced hippocampal neurogenesis.

  15. Kruppel-Like Transcription Factors in the Nervous System: Novel players in neurite outgrowth and axon regeneration

    PubMed Central

    Moore, Darcie L.; Apara, Akintomide; Goldberg, Jeffrey L.

    2011-01-01

    The Krüppel-like family of transcription factors (KLFs) have been widely studied in proliferating cells, though very little is known about their role in post-mitotic cells, such as neurons. We have recently found that the KLFs play a role in regulating intrinsic axon growth ability in retinal ganglion cells (RGCs), a type of central nervous system (CNS) neuron. Previous KLF studies in other cell types suggest that there may be cell-type specific KLF expression patterns, and that their relative expression allows them to compete for binding sites, or to act redundantly to compensate for another’s function. With at least 15 of 17 KLF family members expressed in neurons, it will be important for us to determine how this complex family functions to regulate the intricate gene programs of axon growth and regeneration. By further characterizing the mechanisms of the KLF family in the nervous system, we may better understand how they regulate neurite growth and axon regeneration. PMID:21635952

  16. PTEN deletion enhances survival, neurite outgrowth and function of dopamine neuron grafts to MitoPark mice

    PubMed Central

    Zhang, YaJun; Granholm, Ann-Charlotte; Huh, Kyounghee; Shan, Lufei; Diaz-Ruiz, Oscar; Malik, Nasir; Olson, Lars; Hoffer, Barry J.; Lupica, Carl R.; Hoffman, Alexander F.

    2012-01-01

    Clinical trials in Parkinson’s disease have shown that transplants of embryonic mesencephalic dopamine neurons form new functional connections within the host striatum, but the therapeutic benefits have been highly variable. One obstacle has been poor survival and integration of grafted dopamine neurons. Activation of Akt, a serine/threonine kinase that promotes cell survival and growth, increases the ability of neurons to survive after injury and to regenerate lost neuronal connections. Because the lipid phosphatase, phosphatase and tensin homolog (PTEN) inhibits Akt, we generated a mouse with conditional knock-out of PTEN in dopamine neurons, leading to constitutive expression of Akt in these neurons. Ventral mesencephalic tissue from dopamine phosphatase and tensin homologue knock-out or control animals was then transplanted bilaterally into the dopamine depleted striata of MitoPark mice that express a parkinsonian phenotype because of severe respiratory chain dysfunction in dopamine neurons. After transplantation into MitoPark mice, PTEN-deficient dopamine neurons were less susceptible to cell death, and exhibited a more extensive pattern of fibre outgrowth compared to control grafts. Voltammetric measurements demonstrated that dopamine release and reuptake were significantly increased in the striata of animals receiving dopamine PTEN knock-out transplants. These animals also displayed enhanced spontaneous and drug-induced locomotor activity, relative to control transplanted MitoPark mice. Our results suggest that disinhibition of the Akt-signalling pathway may provide a valuable strategy to enhance survival, function and integration of grafted dopamine neurons within the host striatum and, more generally, to improve survival and integration of different forms of neural grafts. PMID:22961549

  17. The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells.

    PubMed

    Mobarak, C D; Anderson, K D; Morin, M; Beckel-Mitchener, A; Rogers, S L; Furneaux, H; King, P; Perrone-Bizzozero, N I

    2000-09-01

    The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.

  18. Prenatal low-dose methylmercury exposure impairs neurite outgrowth and synaptic protein expression and suppresses TrkA pathway activity and eEF1A1 expression in the rat cerebellum.

    PubMed

    Fujimura, Masatake; Usuki, Fusako; Cheng, Jinping; Zhao, Wenchang

    2016-05-01

    Methylmercury (MeHg) is a highly neurotoxic environmental chemical that can cause developmental impairments. Human fetuses and neonates are particularly susceptible to MeHg toxicity; however, the mechanisms governing its effects in the developing brain are unclear. In the present study, we investigated the effects of prenatal and lactational MeHg exposure on the developing cerebellum in rats. We demonstrated that exposure to 5ppm MeHg decreased postnatal expression of pre- and postsynaptic proteins, suggesting an impairment in synaptic development. MeHg exposure also reduced neurite outgrowth, as shown by a decrease in the expression of the neurite marker neurofilament H. These changes were not observed in rats exposed to 1ppm MeHg. In order to define the underlying mechanism, we investigated the effects of MeHg exposure on the tropomyosin receptor kinase (Trk) A pathway, which plays important roles in neuronal differentiation and synapse formation. We demonstrated suppression of the TrkA pathway on gestation day 20 in rats exposed to 5ppm MeHg. In addition, down-regulation of eukaryotic elongation factor 1A1 (eEF1A1) was observed on postnatal day 1. eEF1A1 knockdown in differentiating PC12 cells impaired neurite outgrowth and synaptic protein expression, similar to the results of MeHg exposure in the cerebellum. These results suggest that suppression of the TrkA pathway and subsequent decreases in eEF1A1 expression induced by prenatal exposure to MeHg may lead to reduced neurite outgrowth and synaptic protein expression in the developing cerebellum.

  19. Sucrose is an early modulator of the key hormonal mechanisms controlling bud outgrowth in Rosa hybrida

    PubMed Central

    Barbier, François; Péron, Thomas; Lecerf, Marion; Perez-Garcia, Maria-Dolores; Barrière, Quentin; Rolčík, Jakub; Boutet-Mercey, Stéphanie; Citerne, Sylvie; Lemoine, Remi; Porcheron, Benoît; Roman, Hanaé; Leduc, Nathalie; Le Gourrierec, José; Bertheloot, Jessica; Sakr, Soulaiman

    2015-01-01

    Sugar has only recently been identified as a key player in triggering bud outgrowth, while hormonal control of bud outgrowth is already well established. To get a better understanding of sugar control, the present study investigated how sugar availability modulates the hormonal network during bud outgrowth in Rosa hybrida. Other plant models, for which mutants are available, were used when necessary. Buds were grown in vitro to manipulate available sugars. The temporal patterns of the hormonal regulatory network were assessed in parallel with bud outgrowth dynamics. Sucrose determined bud entrance into sustained growth in a concentration-dependent manner. Sustained growth was accompanied by sustained auxin production in buds, and sustained auxin export in a DR5::GUS-expressing pea line. Several events occurred ahead of sucrose-stimulated bud outgrowth. Sucrose upregulated early auxin synthesis genes (RhTAR1, RhYUC1) and the auxin efflux carrier gene RhPIN1, and promoted PIN1 abundance at the plasma membrane in a pPIN1::PIN1-GFP-expressing tomato line. Sucrose downregulated both RwMAX2, involved in the strigolactone-transduction pathway, and RhBRC1, a repressor of branching, at an early stage. The presence of sucrose also increased stem cytokinin content, but sucrose-promoted bud outgrowth was not related to that pathway. In these processes, several non-metabolizable sucrose analogues induced sustained bud outgrowth in R. hybrida, Pisum sativum, and Arabidopsis thaliana, suggesting that sucrose was involved in a signalling pathway. In conclusion, we identified potential hormonal candidates for bud outgrowth control by sugar. They are central to future investigations aimed at disentangling the processes that underlie regulation of bud outgrowth by sugar. PMID:25873679

  20. Sucrose is an early modulator of the key hormonal mechanisms controlling bud outgrowth in Rosa hybrida.

    PubMed

    Barbier, François; Péron, Thomas; Lecerf, Marion; Perez-Garcia, Maria-Dolores; Barrière, Quentin; Rolčík, Jakub; Boutet-Mercey, Stéphanie; Citerne, Sylvie; Lemoine, Remi; Porcheron, Benoît; Roman, Hanaé; Leduc, Nathalie; Le Gourrierec, José; Bertheloot, Jessica; Sakr, Soulaiman

    2015-05-01

    Sugar has only recently been identified as a key player in triggering bud outgrowth, while hormonal control of bud outgrowth is already well established. To get a better understanding of sugar control, the present study investigated how sugar availability modulates the hormonal network during bud outgrowth in Rosa hybrida. Other plant models, for which mutants are available, were used when necessary. Buds were grown in vitro to manipulate available sugars. The temporal patterns of the hormonal regulatory network were assessed in parallel with bud outgrowth dynamics. Sucrose determined bud entrance into sustained growth in a concentration-dependent manner. Sustained growth was accompanied by sustained auxin production in buds, and sustained auxin export in a DR5::GUS-expressing pea line. Several events occurred ahead of sucrose-stimulated bud outgrowth. Sucrose upregulated early auxin synthesis genes (RhTAR1, RhYUC1) and the auxin efflux carrier gene RhPIN1, and promoted PIN1 abundance at the plasma membrane in a pPIN1::PIN1-GFP-expressing tomato line. Sucrose downregulated both RwMAX2, involved in the strigolactone-transduction pathway, and RhBRC1, a repressor of branching, at an early stage. The presence of sucrose also increased stem cytokinin content, but sucrose-promoted bud outgrowth was not related to that pathway. In these processes, several non-metabolizable sucrose analogues induced sustained bud outgrowth in R. hybrida, Pisum sativum, and Arabidopsis thaliana, suggesting that sucrose was involved in a signalling pathway. In conclusion, we identified potential hormonal candidates for bud outgrowth control by sugar. They are central to future investigations aimed at disentangling the processes that underlie regulation of bud outgrowth by sugar. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  1. Inhibition of the outgrowth and elongation of neurites from pheochromocytoma cells by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and preventive effects of dimethylsulfoniopropionate in the presence of nerve growth factor.

    PubMed

    Nakajima, Kenji; Minematsu, Masaharu; Miyamoto, Yuuichi

    2008-04-01

    The combined effects of dimethylsulfoniopropionate (DMSP) (10(-3), 10(-4) and 10(-5) M) with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (5 ng/mL) and the nerve growth factor (NGF) (5 ng/mL) on the outgrowth and elongation of neurites from pheochromocytoma (PC12) cells were examined on RPMI medium containing fetal bovine serum and horse serum with penicillin and streptomycin in collagen-coated dishes for 5 d. The growth was higher in increasing order of the DMSP (10(-3) M), MPTP and NGF, the DMSP (10(-5) M), MPTP and NGF, the MPTP and NGF group and the control group up to 3 d, but not in the NGF and the DMSP (10(-4) M), MPTP and NGF groups. The growth in all the experimental groups showed plateaus from days 4 to 5. The appearance of neurites from the cells in all the groups showed maxima on the 3rd day. The administration of NGF significantly stimulated the outgrowth of neurites from the cells, while the supplementation of MPTP noticeably inhibited the appearance of neurites even in the presence of NGF up to 5 d. However, the addition of DMSP (10(-3 )and 10(-4) M) to the latter group completely prevented the inhibition of the MPTP. These facts were significantly supported by the photographs of neurite-bearing cells on the 3rd day and also by the photometric analyses examining the reaction of MPTP to DMSP, NGF or Collagen IV.

  2. Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells

    PubMed Central

    Zhang, Congcong; Helmsing, Saskia; Zagrebelsky, Marta; Schirrmann, Thomas; Marschall, Andrea L. J.; Schüngel, Manuela; Korte, Martin; Hust, Michael; Dübel, Stefan

    2012-01-01

    Background Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies. Results Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells. Conclusion The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells. PMID:22292018

  3. Rac1 and Cdc42 but not RhoA or Rho kinase activities are required for neurite outgrowth induced by the Netrin-1 receptor DCC (deleted in colorectal cancer) in N1E-115 neuroblastoma cells.

    PubMed

    Li, Xiaodong; Saint-Cyr-Proulx, Etienne; Aktories, Klaus; Lamarche-Vane, Nathalie

    2002-04-26

    Netrins are chemotropic guidance cues that attract or repel growing axons during development. DCC (deleted in colorectal cancer), a transmembrane protein that is a receptor for netrin-1, is implicated in mediating both responses. However, the mechanism by which this is achieved remains unclear. Here we report that Rho GTPases are required for embryonic spinal commissural axon outgrowth induced by netrin-1. Using N1E-115 neuroblastoma cells, we found that both Rac1 and Cdc42 activities are required for DCC-induced neurite outgrowth. In contrast, down-regulation of RhoA and its effector Rho kinase stimulates the ability of DCC to induce neurite outgrowth. In Swiss 3T3 fibroblasts, DCC was found to trigger actin reorganization through activation of Rac1 but not Cdc42 or RhoA. We detected that stimulation of DCC receptors with netrin-1 resulted in a 4-fold increase in Rac1 activation. These results implicate the small GTPases Rac1, Cdc42, and RhoA as essential components that participate in signaling the response of axons to netrin-1 during neural development.

  4. Phosphorylation of p85 beta PIX, a Rac/Cdc42-specific guanine nucleotide exchange factor, via the Ras/ERK/PAK2 pathway is required for basic fibroblast growth factor-induced neurite outgrowth.

    PubMed

    Shin, Eun-Young; Shin, Kyung-Sun; Lee, Chan-Soo; Woo, Kyung-Nam; Quan, Song-Hua; Soung, Nak-Kyun; Kim, Young Gyu; Cha, Choong Ik; Kim, Seung-Ryul; Park, Dongeun; Bokoch, Gary M; Kim, Eung-Gook

    2002-11-15

    Guanine nucleotide exchange factors (GEFs) have been implicated in growth factor-induced neuronal differentiation through the activation of small GTPases. Although phosphorylation of these GEFs is considered an activation mechanism, little is known about the upstream of PAK-interacting exchange factor (PIX), a member of the Dbl family of GEFs. We report here that phosphorylation of p85 betaPIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 betaPIX in basic fibroblast growth factor (bFGF)-induced neurite outgrowth, we established PC12 cell lines that overexpress the fibroblast growth factor receptor-1 in a tetracycline-inducible manner. Treatment with bFGF induces the phosphorylation of p85 betaPIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 betaPIX is inhibited by PD98059, a specific MEK inhibitor, suggesting the involvement of the ERK cascade. PAK2, a major PAK isoform in PC12 cells as well as a binding partner of p85 betaPIX, also functions upstream of p85 betaPIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 betaPIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 betaPIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 betaPIX efficiently blocks PC12 cell neurite outgrowth. Our study defines a novel signaling pathway for bFGF-induced neurite outgrowth that involves activation of the PAK2-p85 betaPIX complex via the ERK cascade and subsequent translocation of this complex.

  5. Microwave-assisted synthesis of 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B-355252): a new potentiator of Nerve Growth Factor (NGF)-induced neurite outgrowth

    PubMed Central

    Williams, Alfred L.; Dandepally, Srinivasa R.; Gilyazova, Nailya; Witherspoon, Sam M; Ibeanu, Gordon

    2010-01-01

    The synthesis of 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B-355252) using a MW-assisted nucleophilic aromatic substitution (SNAr) reaction will be discussed. Utilization of this method allowed for the rapid generation of B-355252 heteroaryl ether core structure in the presence of cesium carbonate in dimethylformamide or tripotassium phosphate in N-methyl-2-pyrrolidone in 94% yield. Evaluation of B-355252 enhancement of nerve growth factor’s ability to stimulate neurite outgrowths was determined using NS-1 cells. PMID:22973068

  6. Activin A secreted by human mesenchymal stem cells induces neuronal development and neurite outgrowth in an in vitro model of Alzheimer's disease: neurogenesis induced by MSCs via activin A.

    PubMed

    Park, Sang Eon; Lee, Jeongmin; Chang, Eun Hyuk; Kim, Jong Hwa; Sung, Ji-Hee; Na, Duk L; Chang, Jong Wook

    2016-08-01

    Alzheimer's disease (AD) is characterized by progressive loss of memory in addition to cortical atrophy. Cortical atrophy in AD brains begins in the parietal and temporal lobes, which are near the subventricular zone (SVZ). The aim of this study was to activate the neurogenesis in the SVZ of AD brains by human mesenchymal stem cells (hMSCs). Neural stem cells (NSCs) were isolated from SVZ of 4-month-old 5XFAD mice. Co-culture of hMSCs with SVZ-derived NSCs from 5XFAD mice induced neuronal development and neurite outgrowth. To examine the inducing factor of neurogenesis, human cytokine array was performed with co-cultured media, and revealed elevated release of activin A from hMSCs. Also, we confirmed that the mRNA levels of activin A and activin receptor in the SVZ of 5XFAD mice were significantly lower than normal mice. Treatment of human recombinant activin A in SVZ-derived NSCs from 5XFAD mice induced neuronal development and neurite outgrowth. These data suggest that use of hMSCs and activin A to recover neurogenesis in future studies of cortical regeneration to treat AD.

  7. Nocturnal hemodialysis is associated with restoration of early-outgrowth endothelial progenitor-like cell function.

    PubMed

    Yuen, Darren A; Kuliszewski, Michael A; Liao, Christine; Rudenko, Dmitriy; Leong-Poi, Howard; Chan, Christopher T

    2011-06-01

    Angiogenesis is a key response to tissue ischemia that may be impaired by uremia. Although early-outgrowth endothelial progenitor-like cells promote angiogenesis in the setting of normal renal function, cells from uremic patients are dysfunctional. When compared with conventional hemodialysis, it was hypothesized that nocturnal hemodialysis would improve the in vivo angiogenic activity of these cells in a well described model of ischemic vascular disease. Early-outgrowth endothelial progenitor-like cells were cultured from healthy controls (n = 5) and age- and gender-matched conventional hemodialysis (12 h/wk, n = 10) and nocturnal hemodialysis (30 to 50 h/wk, n = 9) patients. Cells (5 × 10(5)) or saline were injected into the ischemic hindlimb of athymic nude rats 1 day after left common iliac artery ligation. Although conventional dialysis cell injection had no effect versus saline, nocturnal hemodialysis and healthy control cell injection significantly improved ischemic hindlimb perfusion and capillary density. Nocturnal hemodialysis cell injection was also associated with significant increases in endogenous angiopoietin 1 expression in the ischemic hindlimb compared with saline and conventional dialysis cell injection. In contrast to a conventional dialytic regimen, nocturnal hemodialysis is associated with a significantly improved ability of early-outgrowth endothelial progenitor-like cells to promote angiogenesis and thus restore perfusion in a model of ischemic vascular disease.

  8. Nucleocytoplasmic shuttling of the adapter protein SH2B1beta (SH2-Bbeta) is required for nerve growth factor (NGF)-dependent neurite outgrowth and enhancement of expression of a subset of NGF-responsive genes.

    PubMed

    Maures, Travis J; Chen, Linyi; Carter-Su, Christin

    2009-07-01

    The adapter protein SH2B1 (SH2-B, PSM) is recruited to multiple ligand-activated receptor tyrosine kinases, including the receptors for nerve growth factor (NGF), insulin, and IGF-I as well as the cytokine receptor-associated Janus kinase family kinases. In this study, we examine SH2B1's function in NGF signaling. We show that depleting endogenous SH2B1 using short hairpin RNA against SH2B1 inhibits NGF-dependent neurite outgrowth, but not NGF-mediated phosphorylation of Akt or ERKs 1/2. SH2B1 has been hypothesized to localize and function at the plasma membrane. We identify a nuclear localization signal within SH2B1 and show that it is required for nuclear translocation of SH2B1beta. Mutation of the nuclear localization signal has no effect on NGF-induced activation of TrkA and ERKs 1/2 but prevents SH2B1beta from enhancing NGF-induced neurite outgrowth. Disruption of SH2B1beta nuclear import also prevents SH2B1beta from enhancing NGF-induced transcription of genes important for neuronal differentiation, including those encoding urokinase plasminogen activator receptor, and matrix metalloproteinases 3 and 10. Disruption of SH2B1beta nuclear export by mutation of its nuclear export sequence similarly prevents SH2B1beta enhancement of NGF-induced transcription of those genes. Nuclear translocation of the highly homologous family member SH2B2(APS) was not observed. Together, these data suggest that rather than simply acting as an adapter protein linking signaling proteins to the activated TrkA receptor at the plasma membrane, SH2B1beta must shuttle between the plasma membrane and nucleus to function as a critical component of NGF-induced gene expression and neuronal differentiation.

  9. Overexpression of the monocyte chemokine CCL2 in dorsal root ganglion neurons causes a conditioning-like increase in neurite outgrowth and does so via a STAT3 dependent mechanism.

    PubMed

    Niemi, Jon P; DeFrancesco-Lisowitz, Alicia; Cregg, Jared M; Howarth, Madeline; Zigmond, Richard E

    2016-01-01

    Neuroinflammation plays a critical role in the regeneration of peripheral nerves following axotomy. An injury to the sciatic nerve leads to significant macrophage accumulation in the L5 DRG, an effect not seen when the dorsal root is injured. We recently demonstrated that this accumulation around axotomized cell bodies is necessary for a peripheral conditioning lesion response to occur. Here we asked whether overexpression of the monocyte chemokine CCL2 specifically in DRG neurons of uninjured mice is sufficient to cause macrophage accumulation and to enhance regeneration or whether other injury-derived signals are required. AAV5-EF1α-CCL2 was injected intrathecally, and this injection led to a time-dependent increase in CCL2 mRNA expression and macrophage accumulation in L5 DRG, with a maximal response at 3 weeks post-injection. These changes led to a conditioning-like increase in neurite outgrowth in DRG explant and dissociated cell cultures. This increase in regeneration was dependent upon CCL2 acting through its primary receptor CCR2. When CCL2 was overexpressed in CCR2-/- mice, macrophage accumulation and enhanced regeneration were not observed. To address the mechanism by which CCL2 overexpression enhances regeneration, we tested for elevated expression of regeneration-associated genes in these animals. Surprisingly, we found that CCL2 overexpression led to a selective increase in LIF mRNA and neuronal phosphorylated STAT3 (pSTAT3) in L5 DRGs, with no change in expression seen in other RAGs such as GAP-43. Blockade of STAT3 phosphorylation by each of two different inhibitors prevented the increase in neurite outgrowth. Thus, CCL2 overexpression is sufficient to induce macrophage accumulation in uninjured L5 DRGs and increase the regenerative capacity of DRG neurons via a STAT3-dependent mechanism. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Overexpression of the Monocyte Chemokine CCL2 in Dorsal Root Ganglion Neurons Causes a Conditioning-Like Increase in Neurite Outgrowth and Does So via a STAT3 Dependent Mechanism

    PubMed Central

    Niemi, Jon P.; DeFrancesco-Lisowitz, Alicia; Cregg, Jared; Howarth, Madeline; Zigmond, Richard E.

    2015-01-01

    Neuroinflammation plays a critical role in the regeneration of peripheral nerves following axotomy. An injury to the sciatic nerve leads to significant macrophage accumulation in the L5 DRG, an effect not seen when the dorsal root is injured. We recently demonstrated that this accumulation around axotomized cell bodies is necessary for a peripheral conditioning lesion response to occur. Here we asked whether overexpression of the monocyte chemokine CCL2 specifically in DRG neurons of uninjured mice is sufficient to cause macrophage accumulation and to enhance regeneration or whether other injury-derived signals are required. AAV5-EF1α-CCL2 was injected intrathecally, and this injection led to a time-dependent increase in CCL2 mRNA expression and macrophage accumulation in L5 DRG, with a maximal response at 3 wk post-injection. These changes led to a conditioning-like increase in neurite outgrowth in DRG explant and dissociated cell cultures. This increase in regeneration was dependent upon CCL2 acting through its primary receptor CCR2. When CCL2 was overexpressed in CCR2 −/− mice, macrophage accumulation and enhanced regeneration were not observed. To address the mechanism by which CCL2 overexpression enhances regeneration, we tested for elevated expression of regeneration-associated genes in these animals. Surprisingly, we found that CCL2 overexpression led to a selective increase in LIF mRNA and neuronal phosphorylated STAT3 (pSTAT3) in L5 DRGs, with no change in expression seen in other RAGs such as GAP-43. Blockade of STAT3 phosphorylation by each of two different inhibitors prevented the increase in neurite outgrowth. Thus, CCL2 overexpression is sufficient to induce macrophage accumulation in uninjured L5 DRGs and increase the regenerative capacity of DRG neurons via a STAT3-dependent mechanism. PMID:26431741

  11. A three-dimensional image processing program for accurate, rapid, and semi-automated segmentation of neuronal somata with dense neurite outgrowth

    PubMed Central

    Ross, James D.; Cullen, D. Kacy; Harris, James P.; LaPlaca, Michelle C.; DeWeerth, Stephen P.

    2015-01-01

    Three-dimensional (3-D) image analysis techniques provide a powerful means to rapidly and accurately assess complex morphological and functional interactions between neural cells. Current software-based identification methods of neural cells generally fall into two applications: (1) segmentation of cell nuclei in high-density constructs or (2) tracing of cell neurites in single cell investigations. We have developed novel methodologies to permit the systematic identification of populations of neuronal somata possessing rich morphological detail and dense neurite arborization throughout thick tissue or 3-D in vitro constructs. The image analysis incorporates several novel automated features for the discrimination of neurites and somata by initially classifying features in 2-D and merging these classifications into 3-D objects; the 3-D reconstructions automatically identify and adjust for over and under segmentation errors. Additionally, the platform provides for software-assisted error corrections to further minimize error. These features attain very accurate cell boundary identifications to handle a wide range of morphological complexities. We validated these tools using confocal z-stacks from thick 3-D neural constructs where neuronal somata had varying degrees of neurite arborization and complexity, achieving an accuracy of ≥95%. We demonstrated the robustness of these algorithms in a more complex arena through the automated segmentation of neural cells in ex vivo brain slices. These novel methods surpass previous techniques by improving the robustness and accuracy by: (1) the ability to process neurites and somata, (2) bidirectional segmentation correction, and (3) validation via software-assisted user input. This 3-D image analysis platform provides valuable tools for the unbiased analysis of neural tissue or tissue surrogates within a 3-D context, appropriate for the study of multi-dimensional cell-cell and cell-extracellular matrix interactions. PMID

  12. L- and T-type calcium channel blockers protect against the inhibitory effects of mipafox on neurite outgrowth and plasticity-related proteins in SH-SY5Y cells.

    PubMed

    Fernandes, Laís Silva; Dos Santos, Neife Aparecida G; Emerick, Guilherme Luz; Santos, Antonio Cardozo Dos

    2017-09-01

    Some organophosphorus compounds (OP), including the pesticide mipafox, produce late onset distal axonal degeneration, known as organophosphorus-induced delayed neuropathy (OPIDN). The underlying mechanism involves irreversible inhibition of neuropathy target esterase (NTE) activity, elevated intracellular calcium levels, increased activity of calcium-activated proteases and impaired neuritogenesis. Voltage-gated calcium channels (VGCC) appear to play a role in several neurologic disorders, including OPIDN. Therefore, this study aimed to examine and compare the neuroprotective effects of T-type (amiloride) and L-type (nimodipine) VGCC blockers induced by the inhibitory actions of mipafox on neurite outgrowth and axonal proteins of retinoic-acid-stimulated SH-SY5Y human neuroblastoma cells, a neuronal model widely employed to determine the neurotoxicity attributed to OP. Both nimodipine and amiloride significantly blocked augmentation of intracellular calcium levels and activity of calpains, as well as decreased neurite length, number of differentiated cells, and lowered concentrations of growth-associated protein 43 (GAP-43) and synapsin induced by mipafox. Only nimodipine inhibited reduction of synaptophysin levels produced by mipafox. These findings demonstrate a role for calcium and VGCC in the impairment of neuronal plasticity mediated by mipafox. Data also demonstrated the neuroprotective potential of T-type and L-type VGCC blockers to inhibit OP-mediated actions, which may be beneficial to counteract cases of pesticide poisoning.

  13. Induction of neurite outgrowth by interleukin-6 is accompanied by activation of Stat3 signaling pathway in a variant PC12 cell (E2) line.

    PubMed

    Wu, Y Y; Bradshaw, R A

    1996-05-31

    PC12-E2 cells, a stable variant subcloned from native cell populations, produce neurites in a rapid, transcription-independent manner upon exposure to nerve growth factor (NGF) or basic fibroblast growth factor (bFGF). They also give a similar morphological response to interleukin-6 (IL-6), which is, however, transcription-dependent and with a slower onset, a phenomenon basically not observed in native PC12 cells. The response profile of PC12-E2 cells to NGF and bFGF is similar to that observed for native PC12 cells pre-exposed (primed) to NGF, and such cells also respond to IL-6 in a fashion indistinguishable from PC12-E2 cells. Mechanistically, NGF and bFGF induce a sustained phosphorylation and activation of ERK1 and ERK2 in both cells, while IL-6 produces only a transient and weak tyrosine phosphorylation. However, it does stimulate a prolonged and biphasic tyrosine phosphorylation and nuclear translocation of Stat3 (signal transducers and activators of transcription 3; at least 24 h) and, to a lesser extent, Stat1. Gel shift and supershift analyses confirm that IL-6 predominantly activates Stat3 (and some Stat1) and stimulates sis-inducible element binding activity. Other members of the same cytokine subfamily, including ciliary neurotrophic factor and leukemia inhibitory factor, also cause a transient initial phase of tyrosine phosphorylation and activation of Stat1 and Stat3 (up to 1 h) but fail to stimulate a second phase of response and do not produce significant neurites. These results suggest that sustained signaling of either STAT or ERK pathways in PC12-E2 cells leads to induction of neuronal differentiation. However, only the latter is effective in native PC12 cells as the activation of Stat3 and Stat1 in native PC12 cells by IL-6 fails to induce neuronal differentiation. Thus, the response of PC12-E2 cells to IL-6 suggests the constitutive expression of a required factor(s) for differentiation, that is induced in native PC12 cells by NGF or b

  14. 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.

  15. Targeting RPTPσ with lentiviral shRNA promotes neurites outgrowth of cortical neurons and improves functional recovery in a rat spinal cord contusion model.

    PubMed

    Zhou, Heng-Xing; Li, Xue-Ying; Li, Fu-Yuan; Liu, Chang; Liang, Zhi-Pin; Liu, Shen; Zhang, Bin; Wang, Tian-Yi; Chu, Tian-Ci; Lu, Lu; Ning, Guang-Zhi; Kong, Xiao-Hong; Feng, Shi-Qing

    2014-10-24

    After spinal cord injury (SCI), the rapidly upregulated chondroitin sulfate proteoglycans (CSPGs), the prominent chemical constituents and main repulsive factors of the glial scar, play an important role in the extremely limited ability to regenerate in adult mammals. Although many methods to overcome the inhibition have been tested, no successful method with clinical feasibility has been devised to date. It was recently discovered that receptor protein tyrosine phosphatase sigma (RPTPσ) is a functional receptor for CSPGs-mediated inhibition. In view of the potential clinical application of RNA interference (RNAi), here we investigated whether silencing RPTPσ via lentivirus-mediated RNA interference can promote axon regeneration and functional recovery after SCI. Neurites of primary rat cerebral cortical neurons with depleted RPTPσ exhibited a significant enhancement in elongation and crossing ability when they encountered CSPGs in vitro. A contusion model of spinal cord injury in Wistar rats (the New York University (NYU) impactor) was used for in vivo experiments. Local injection of lentivirus encoding RPTPσ shRNA at the lesion site promoted axon regeneration and synapse formation, but did not affect the scar formation. Meanwhile, in vivo functional recovery (motor and sensory) was also enhanced after RPTPσ depletion. Therefore, strategies directed at silencing RPTPσ by RNAi may prove to be a beneficial, efficient and valuable approach for the treatment of SCI. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Scorpion venom heat-resistant peptide (SVHRP) enhances neurogenesis and neurite outgrowth of immature neurons in adult mice by up-regulating brain-derived neurotrophic factor (BDNF).

    PubMed

    Wang, Tao; Wang, Shi-Wei; Zhang, Yue; Wu, Xue-Fei; Peng, Yan; Cao, Zhen; Ge, Bi-Ying; Wang, Xi; Wu, Qiong; Lin, Jin-Tao; Zhang, Wan-Qin; Li, Shao; Zhao, Jie

    2014-01-01

    Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Although scorpions and their venom have been used in Traditional Chinese Medicine (TCM) to treat chronic neurological disorders, the underlying mechanisms of these treatments remain unknown. We applied SVHRP in vitro and in vivo to understand its effects on the neurogenesis and maturation of adult immature neurons and explore associated molecular mechanisms. SVHRP administration increased the number of 5-bromo-2'-dexoxyuridine (BrdU)-positive cells, BrdU-positive/neuron-specific nuclear protein (NeuN)-positive neurons, and polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive immature neurons in the subventricular zone (SVZ) and subgranular zone (SGZ) of hippocampus. Furthermore immature neurons incubated with SVHRP-pretreated astrocyte-conditioned medium exhibited significantly increased neurite length compared with those incubated with normal astrocyte-conditioned medium. This neurotrophic effect was further confirmed in vivo by detecting an increased average single area and whole area of immature neurons in the SGZ, SVZ and olfactory bulb (OB) in the adult mouse brain. In contrast to normal astrocyte-conditioned medium, higher concentrations of brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) was detected in the conditioned medium of SVHRP-pretreated astrocytes, and blocking BDNF using anti-BDNF antibodies eliminated these SVHRP-dependent neurotrophic effects. In SVHRP treated mouse brain, more glial fibrillary acidic protein (GFAP)-positive cells were detected. Furthermore, immunohistochemistry revealed increased numbers of GFAP/BDNF double-positive cells, which agrees with the observed changes in the culture system. This paper describes novel effects of scorpion venom-originated peptide on the stem cells and suggests the potential therapeutic values of SVHRP.

  17. Laminin receptors for neurite formation

    SciTech Connect

    Kleinman, H.K.; Ogle, R.C.; Cannon, F.B.; Little, C.D.; Sweeney, T.M.; Luckenbill-Edds, L.

    1988-02-01

    Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. The authors report here that /sup 125/I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein was found in a variety of epithelial cell lines and in brain, whereas the 180-kDa protein was neural specific. Antibodies prepared against the 110-kDa and 180-kDa proteins inhibited neurite outgrowth induced by the neurite-promoting domain of laminin, whereas antibodies to the 67-kDa laminin receptor had no effect on neurite outgrowth. They conclude that neuronal cells have multiple cell-surface laminin receptors and that the 110-kDa and 180-kDa proteins are involved in neurite formation.

  18. Angiogenic potential of early and late outgrowth endothelial progenitor cells is dependent on the time of emergence.

    PubMed

    Minami, Yoshiyasu; Nakajima, Toshiaki; Ikutomi, Masayasu; Morita, Toshihiro; Komuro, Issei; Sata, Masataka; Sahara, Makoto

    2015-01-01

    Recent studies have suggested that late-outgrowth endothelial progenitor cells (EPCs) derived from human peripheral blood mononuclear cells (hPBMNCs) might have higher angiogenic potential than classically-defined early-outgrowth EPCs (EOCs). However, it still remains unclear which of "so-called" EPC subpopulations defined in a variety of ways has the highest angiogenic potential. We classified hPBMNC-derived EPC subpopulations by the time of their emergence in culture. EOCs were defined as attached cells on culture days 3-7. Late-outgrowth EPCs, defined as the cell forming colonies with cobblestone appearance since day 10, were further classified as follows: "moderate"-outgrowth EPCs (MOCs) emerging on days 10-16, "late"-outgrowth EPCs (LOCs) on days 17-23, and "very late"-outgrowth EPCs (VOCs) on days 24-30. Flow cytometry analyses showed the clear differences of hematopoietic/endothelial markers between EOC (CD31(+)VE-cadherin(-)CD34(-)CD14(+)CD45(+)) and LOC (CD31(+)VE-cadherin(+)CD34(+)CD14(-)CD45(-)). We found that LOCs had the highest proliferation and tube formation capabilities in vitro along with the highest expression of angiogenic genes including KDR and eNOS. To investigate the in vivo therapeutic efficacies, each EPC subpopulation was intravenously transplanted into immunocompromised mice (total 4 × 10(5) cells) after unilateral hindlimb ischemia surgery. The LOC-treated mice exhibited significantly-enhanced blood flow recovery (flow ratios of ischemic/non-ischemic leg: 0.99±0.02 [LOC group] versus 0.67 ± 0.07 to 0.78 ± 0.09 [other groups]; P < 0.05) and augmented capillary collateral formation in ischemic leg, which were attributable to their direct engraftment into host angiogenic vessels (approximately 10%) and paracrine effects. hPBMNC-derived late-outgrowth EPCs emerging on culture days 17-23 are superior to other EPC subpopulations with regard to therapeutic angiogenic potential. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. Effect of smoking cessation on the number and adhesive properties of early outgrowth endothelial progenitor cells.

    PubMed

    Puls, Miriam; Schroeter, Marco R; Steier, Jasmin; Stijohann, Lena; Hasenfuss, Gerd; Konstantinides, Stavros; Schäfer, Katrin

    2011-10-06

    Endothelial progenitor cells participate in angiogenesis and vascular repair, and cardiovascular risk factors may reduce their numbers or impair their functional properties. Cigarette smoking is a leading cause of preventable cardiovascular death, however, the functional properties of these cells before and after discontinuation of tobacco use have not been systematically analyzed. We examined changes in the number and function of early outgrowth endothelial progenitor cells (EPC), isolated from individuals (n=144; mean age, 47.8 ± 12.0 years; 43% males; more than 50% with additional cardiovascular risk factors or disease) who successfully completed a 5-week smoking cessation (SC) programme. SC significantly reduced total white blood cell count (WBC; P<0.0001), plasma LDL cholesterol (P=0.0002) and fibrinogen (P<0.0001) levels, but did not alter the number of circulating CD34(+), VEGFR2(+) or CD34(+), CD133(+) cells (P=0.14 and 0.57, respectively). Fewer acLDL(+), lectin(+) cells could be expanded from peripheral blood mononuclear cells in comparison to baseline (P<0.001). Furthermore, SC was associated with reduced EPC adhesion to fibronectin (P<0.001) or TNFα-activated endothelial cells (P=0.003), and a diminished incorporation of EPC into endothelial cell networks (P=0.035). Mechanistically, significantly reduced β1- and β2-integrin expression (P<0.001 and 0.007) and lower contents of intracellular reactive oxygen species (P<0.007) were detected in EPC following SC, in addition to reduced plasma asymmetric dimethyl-L-arginine (ADMA) levels (P=0.0003). Our findings suggest that the oxidative and inflammatory stress reduction associated with smoking cessation impair the adhesiveness of monocyte-derived EPC. Copyright © 2010. Published by Elsevier Ireland Ltd.

  20. Early Outgrowth Cells Release Soluble Endocrine Antifibrotic Factors That Reduce Progressive Organ Fibrosis

    PubMed Central

    Yuen, Darren A.; Connelly, Kim A.; Zhang, Yanling; Advani, Suzanne L.; Thai, Kerri; Kabir, Golam; Kepecs, David; Spring, Christopher; Smith, Christopher; Batruch, Ihor; Kosanam, Hari; Advani, Andrew; Diamandis, Eleftherios; Marsden, Philip A.; Gilbert, Richard E.

    2017-01-01

    Adult bone marrow-derived cells can improve organ function in chronic disease models, ostensibly by the release of paracrine factors. It has, however, been difficult to reconcile this prevailing paradigm with the lack of cell retention within injured organs and their rapid migration to the reticuloendothelial system. Here, we provide evidence that the salutary antifibrotic effects of bone marrow-derived early outgrowth cells (EOCs) are more consistent with an endocrine mode of action, demonstrating not only the presence of antifibrotic factors in the plasma of EOC-treated rats but also that EOC conditioned medium (EOC-CM) potently attenuates both TGF-β- and angiotensin II-induced fibroblast collagen production in vitro. To examine the therapeutic relevance of these findings in vivo, 5/6 subtotally nephrectomized rats, a model of chronic kidney and heart failure characterized by progressive fibrosis of both organs, were randomized to receive i.v. injections of EOC-CM, unconditioned medium, or 106 EOCs. Rats that received unconditioned medium developed severe kidney injury with cardiac diastolic dysfunction. In comparison, EOC-CM-treated rats demonstrated substantially improved renal and cardiac function and structure, mimicking the changes found in EOC-treated animals. Mass spectrometric analysis of EOC-CM identified proteins that regulate cellular functions implicated in fibrosis. These results indicate that EOCs secrete soluble factor(s) with highly potent antifibrotic activity, that when injected intravenously replicate the salutary effects of the cells themselves. Together, these findings suggest that an endocrine mode of action may underlie the effectiveness of cell therapy in certain settings and portend the possibility for systemic delivery of cell-free therapy. PMID:23922321

  1. Analysis of compaction, allocation, and outgrowth in the early mouse embryo

    SciTech Connect

    Sutherland, A.E.

    1988-01-01

    Examination of the process of compaction by cinemicrography, and by the use of cytoskeleton-disrupting agents showed it to be a variable, progressive event, dependent on microfilaments both for cell flattering and for polarization of surface microvilli. Examination of the subsequent cleavage division by cinemicrography revealed that there are three major planes of division, and made possible the determination of their relative frequencies during fourth cleavage. Attachment and trophoblast outgrowth on substrates of extracellular matrix molecules was found to involve a group of 140 kD glycoproteins. Addition to the culture medium of a synthetic peptide containing the Arg-Gly-Asp tripeptide cell recognition sequence of fibronectin inhibited trophoblasts outgrowth, but not attachment, on both laminin and fibronectin. Immunoprecipitation of /sup 125/I surface-labeled embryos revealed that the 140 kD glycoproteins are exposed on the surfaces of embryos during outgrowth formation, but are not detectable immediately after hatching. Immunofluorescence experiments showed that the 140 kD glycoproteins and the cytoskeletal proteins vinculin and talin are enriched on the cell processes and ventral surfaces of trophoblasts cells in embryo outgrowths, in patterns similar to those seen in fibroblasts, and consistent with their role in adhesion of the trophoblasts cells to the substratum.

  2. Inhibition of neurite outgrowth in differentiating mouse N2a neuroblastoma cells by phenyl saligenin phosphate: effects on MAP kinase (ERK 1/2) activation, neurofilament heavy chain phosphorylation and neuropathy target esterase activity.

    PubMed

    Hargreaves, Alan J; Fowler, Maxine J; Sachana, Magdalini; Flaskos, John; Bountouri, Mary; Coutts, Ian C; Glynn, Paul; Harris, Wayne; Graham McLean, W

    2006-04-14

    Sub-lethal concentrations of the organophosphate phenyl saligenin phosphate (PSP) inhibited the outgrowth of axon-like processes in differentiating mouse N2a neuroblastoma cells (IC(50) 2.5 microM). A transient rise in the phosphorylation state of neurofilament heavy chain (NFH) was detected on Western blots of cell extracts treated with 2.5 microM PSP for 4 h compared to untreated controls, as determined by a relative increase in reactivity with monoclonal antibody Ta51 (anti-phosphorylated NFH) compared to N52 (anti-total NFH). However, cross-reactivity of PSP-treated cell extracts was lower than that of untreated controls after 24 h exposure, as indicated by decreased reactivity with both antibodies. Indirect immunofluorescence analysis with these antibodies revealed the appearance of neurofilament aggregates in the cell bodies of treated cells and reduced axonal staining compared to controls. By contrast, there was no significant change in reactivity with anti-alpha-tubulin antibody B512 at either time point. The activation state of the MAP kinase ERK 1/2 increased significantly after PSP treatment compared to controls, particularly at 4 h, as indicated by increased reactivity with monoclonal antibody E-4 (anti-phosphorylated MAP kinase) but not with polyclonal antibody K-23 (anti-total MAP kinase). The observed early changes were concomitant with almost complete inhibition of the activity of neuropathy target esterase (NTE), one of the proposed early molecular targets in organophosphate-induced delayed neuropathy (OPIDN).

  3. Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

    PubMed

    Kawamura, Kazuhiro; Chen, Yuan; Shu, Yimin; Cheng, Yuan; Qiao, Jie; Behr, Barry; Pera, Renee A Reijo; Hsueh, Aaron J W

    2012-01-01

    Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility

  4. Neurulation and neurite extension require the zinc transporter ZIP12 (slc39a12)

    PubMed Central

    Chowanadisai, Winyoo; Graham, David M.; Keen, Carl L.; Rucker, Robert B.; Messerli, Mark A.

    2013-01-01

    Zn2+ is required for many aspects of neuronal structure and function. However, the regulation of Zn2+ in the nervous system remains poorly understood. Systematic analysis of tissue-profiling microarray data showed that the zinc transporter ZIP12 (slc39a12) is highly expressed in the human brain. In the work reported here, we confirmed that ZIP12 is a Zn2+ uptake transporter with a conserved pattern of high expression in the mouse and Xenopus nervous system. Mouse neurons and Neuro-2a cells produce fewer and shorter neurites after ZIP12 knockdown without affecting cell viability. Zn2+ chelation or loading in cells to alter Zn2+ availability respectively mimicked or reduced the effects of ZIP12 knockdown on neurite outgrowth. ZIP12 knockdown reduces cAMP response element-binding protein activation and phosphorylation at serine 133, which is a critical pathway for neuronal differentiation. Constitutive cAMP response element-binding protein activation restores impairments in neurite outgrowth caused by Zn2+ chelation or ZIP12 knockdown. ZIP12 knockdown also reduces tubulin polymerization and increases sensitivity to nocodazole following neurite outgrowth. We find that ZIP12 is expressed during neurulation and early nervous system development in Xenopus tropicalis, where ZIP12 antisense morpholino knockdown impairs neural tube closure and arrests development during neurulation with concomitant reduction in tubulin polymerization in the neural plate. This study identifies a Zn2+ transporter that is specifically required for nervous system development and provides tangible links between Zn2+, neurulation, and neuronal differentiation. PMID:23716681

  5. The hormone melatonin stimulates renoprotective effects of "early outgrowth" endothelial progenitor cells in acute ischemic kidney injury.

    PubMed

    Patschan, D; Hildebrandt, A; Rinneburger, J; Wessels, J T; Patschan, S; Becker, J U; Henze, E; Krüger, A; Müller, G A

    2012-05-15

    Endothelial progenitor cells (EPCs) protect the kidney from acute ischemic injury. The aim of this study was to analyze whether pretreatment of murine "early outgrowth" EPCs (eEPCs) with the hormone melatonin increases the cells' renoprotective effects in the setting of murine acute ischemic renal failure. Male (8-12 wk old) C57Bl/6N mice were subjected to unilateral ischemia-reperfusion injury postuninephrectomy (40 min). Postischemic animals were injected with either 0.5×10(6) untreated syngeneic murine eEPCs or with cells, pretreated with melatonin for 1 h. Injections were performed shortly after reperfusion of the kidney. While animals injected with untreated cells developed acute renal failure, eEPC pretreatment with melatonin dramatically improved renoprotective actions of the cells. These effects were completely reversed after cell pretreatment with melatonin and the MT-1/-2 antagonist luzindole. In vitro analysis revealed that melatonin reduced the amount of tumor growth factor-β-induced eEPC apoptosis/necrosis. Secretion of vascular endothelial growth factor by the cells was markedly stimulated by the hormone. In addition, migratory activity of eEPCs was enhanced by melatonin and supernatant from melatonin-treated eEPCs stimulated migration of cultured mature endothelial cells. In summary, melatonin was identified as a new agonist of eEPCs in acute ischemic kidney injury.

  6. Cerebral ischaemia and matrix metalloproteinase-9 modulate the angiogenic function of early and late outgrowth endothelial progenitor cells.

    PubMed

    Morancho, Anna; Hernández-Guillamon, Mar; Boada, Cristina; Barceló, Verónica; Giralt, Dolors; Ortega, Laura; Montaner, Joan; Rosell, Anna

    2013-12-01

    The enhancement of endogenous angiogenesis after stroke will be critical in neurorepair therapies where endothelial progenitor cells (EPCs) might be key players. Our aim was to determine the influence of cerebral ischaemia and the role of matrix metalloproteinase-9 (MMP-9) on the angiogenic function of EPCs. Permanent focal cerebral ischaemia was induced by middle cerebral artery (MCA) occlusion in MMP-9/knockout (MMP-9/KO) and wild-type (WT) mice. EPCs were obtained for cell counting after ischaemia (6 and 24 hrs) and in control animals. Matrigel(™) assays and time-lapse imaging were conducted to monitor angiogenic function of WT and MMP9-deficient EPCs or after treatment with MMP-9 inhibitors. Focal cerebral ischaemia increased the number of early EPCs, while MMP-9 deficiency decreased their number in non-ischaemic mice and delayed their release after ischaemia. Late outgrowth endothelial cells (OECs) from ischaemic mice shaped more vessel structures than controls, while MMP-9 deficiency reduced the angiogenic abilities of OECs to form vascular networks, in vitro. Treatment with the MMP inhibitor GM6001 and the specific MMP-9 inhibitor I also decreased the number of vessel structures shaped by both human and mouse WT OECs, while exogenous MMP-9 could not revert the impaired angiogenic function in MMP-9/KO OECs. Finally, time-lapse imaging showed that the extension of vascular networks was influenced by cerebral ischaemia and MMP-9 deficiency early during the vascular network formation followed by a dynamic vessel remodelling. We conclude that focal cerebral ischaemia triggers the angiogenic responses of EPCs, while MMP-9 plays a key role in the formation of vascular networks by EPCs. © 2013 The Authors. Journal of Cellular and Molecular Medicine Published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  7. Cerebral ischaemia and matrix metalloproteinase-9 modulate the angiogenic function of early and late outgrowth endothelial progenitor cells

    PubMed Central

    Morancho, Anna; Hernández-Guillamon, Mar; Boada, Cristina; Barceló, Verónica; Giralt, Dolors; Ortega, Laura; Montaner, Joan; Rosell, Anna

    2013-01-01

    The enhancement of endogenous angiogenesis after stroke will be critical in neurorepair therapies where endothelial progenitor cells (EPCs) might be key players. Our aim was to determine the influence of cerebral ischaemia and the role of matrix metalloproteinase-9 (MMP-9) on the angiogenic function of EPCs. Permanent focal cerebral ischaemia was induced by middle cerebral artery (MCA) occlusion in MMP-9/knockout (MMP-9/KO) and wild-type (WT) mice. EPCs were obtained for cell counting after ischaemia (6 and 24 hrs) and in control animals. Matrigel™ assays and time-lapse imaging were conducted to monitor angiogenic function of WT and MMP9-deficient EPCs or after treatment with MMP-9 inhibitors. Focal cerebral ischaemia increased the number of early EPCs, while MMP-9 deficiency decreased their number in non-ischaemic mice and delayed their release after ischaemia. Late outgrowth endothelial cells (OECs) from ischaemic mice shaped more vessel structures than controls, while MMP-9 deficiency reduced the angiogenic abilities of OECs to form vascular networks, in vitro. Treatment with the MMP inhibitor GM6001 and the specific MMP-9 inhibitor I also decreased the number of vessel structures shaped by both human and mouse WT OECs, while exogenous MMP-9 could not revert the impaired angiogenic function in MMP-9/KO OECs. Finally, time-lapse imaging showed that the extension of vascular networks was influenced by cerebral ischaemia and MMP-9 deficiency early during the vascular network formation followed by a dynamic vessel remodelling. We conclude that focal cerebral ischaemia triggers the angiogenic responses of EPCs, while MMP-9 plays a key role in the formation of vascular networks by EPCs. PMID:23945132

  8. Early postweaning social isolation but not environmental enrichment modifies vermal Purkinje cell dendritic outgrowth in rats.

    PubMed

    Pascual, Rodrigo; Bustamante, Carlos

    2013-01-01

    In the present study, we analyzed the effects of enriched, social and isolated experiences on vermal Purkinje cell of the rat, together with anxiety-like behavior in the elevated-plus maze. Sprague-Dawley male rats were randomly submitted to either enriched, social, or isolated environments during the early postweaning period (postnatal days 22-32) and were then behaviorally evaluated in the elevated-plus maze and euthanized for histological analysis. Vermal Purkinje cells (sub-lobules VIa and VIb) were sampled, drawn under camera lucida and morphometrically assessed using the Sholl's concentric ring method. Data obtained indicate that environmental enrichment did not significantly modify the Purkinje cell dendritic branching. On the contrary, Purkinje cell of animals reared in social isolation exhibited a significant reduction in dendritic arborization, which was closely associated with anxiety-like behaviors. The data obtained indicate that, although environmental stimulation in normal animals does not produce significant changes in vermal Purkinje cell dendritic arborization, these cells are vulnerable to early stressful experiences, which is in close association with anxiety-like behaviors.

  9. The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells.

    PubMed

    Talman, Virpi; Amadio, Marialaura; Osera, Cecilia; Sorvari, Salla; Boije Af Gennäs, Gustav; Yli-Kauhaluoma, Jari; Rossi, Daniela; Govoni, Stefano; Collina, Simona; Ekokoski, Elina; Tuominen, Raimo K; Pascale, Alessia

    2013-07-01

    Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20μM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10μM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that

  10. 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.

  11. Conditioned Medium from Early-Outgrowth Bone Marrow Cells Is Retinal Protective in Experimental Model of Diabetes

    PubMed Central

    Duarte, Diego A.; Papadimitriou, Alexandros; Gilbert, Richard E.; Thai, Kerri; Zhang, Yanling; Rosales, Mariana A. B.; Lopes de Faria, José B.; Lopes de Faria, Jacqueline M.

    2016-01-01

    Bone marrow-derived cells were demonstrated to improve organ function, but the lack of cell retention within injured organs suggests that the protective effects are due to factors released by the cells. Herein, we tested cell therapy using early outgrowth cells (EOCs) or their conditioned media (CM) to protect the retina of diabetic animal models (type 1 and type 2) and assessed the mechanisms by in vitro study. Control and diabetic (db/db) mice (8 weeks of age) were randomized to receive a unique intravenous injection of 5×105EOCs or 0.25 ml thrice weekly tail-vein injections of 10x concentrated CM and Wystar Kyoto rats rendered diabetic were randomized to receive 0.50 ml thrice weekly tail-vein injections of 10x concentrated CM. Four weeks later, the animals were euthanized and the eyes were enucleated. Rat retinal Müller cells (rMCs) were exposed for 24 h to high glucose (HG), combined or not with EOC-conditioned medium (EOC-CM) from db/m EOC cultures. Diabetic animals showed increase in diabetic retinopathy (DR) and oxidative damage markers; the treatment with EOCs or CM infusions significantly reduced this damage and re-established the retinal function. In rMCs exposed to diabetic milieu conditions (HG), the presence of EOC-CM reduced reactive oxygen species production by modulating the NADPH-oxidase 4 system, thus upregulating SIRT1 activity and deacetylating Lys-310-p65-NFκB, decreasing GFAP and VEGF expressions. The antioxidant capacity of EOC-CM led to the prevention of carbonylation and nitrosylation posttranslational modifications on the SIRT1 molecule, preserving its activity. The pivotal role of SIRT1 on the mode of action of EOCs or their CM was also demonstrated on diabetic retina. These findings suggest that EOCs are effective as a form of systemic delivery for preventing the early molecular markers of DR and its conditioned medium is equally protective revealing a novel possibility for cell-free therapy for the treatment of DR. PMID:26836609

  12. Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics.

    PubMed

    Speranza, Luisa; Giuliano, Teresa; Volpicelli, Floriana; De Stefano, M Egle; Lombardi, Loredana; Chambery, Angela; Lacivita, Enza; Leopoldo, Marcello; Bellenchi, Gian C; di Porzio, Umberto; Crispino, Marianna; Perrone-Capano, Carla

    2015-01-01

    Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

  13. Extracellular matrix allows PC12 neurite elongation in the absence of microtubules.

    PubMed

    Lamoureux, P; Steel, V L; Regal, C; Adgate, L; Buxbaum, R E; Heidemann, S R

    1990-01-01

    Several groups have shown that PC12 will extend microtubule-containing neurites on extracellular matrix (ECM) with no lag period in the absence of nerve growth factor. This is in contrast to nerve growth factor (NGF)-induced neurite outgrowth that occurs with a lag period of several days. During this lag period, increased synthesis or activation of assembly-promoting microtubule-associated proteins (MAPs) occurs and is apparently required for neurite extension. We investigated the growth and microtubule (MT) content of PC12 neurites grown on ECM in the presence or absence of inhibitors of neurite outgrowth. On ECM, neurites of cells with or without prior exposure to NGF contain a normal density of MTs, but frequently contain unusual loops of MTs in their termini that may indicate increased MT assembly. On ECM, neurites extend from PC12 cells in the presence of 10 microM LiCl at significantly higher frequency than on polylysine. On other substrates, LiCl inhibits neurite outgrowth, apparently by inhibiting phosphorylation of particular MAPs (Burstein, D. E., P. J. Seeley, and L. A. Greene. 1985. J. Cell Biol. 101:862-870). Although 35-45% of 60 Li(+)-neurites examined were found to contain a normal array of MTs, 25-30% were found to have a MT density approximately 15% of normal. The remaining 30% of these neurites were found to be nearly devoid of MTs, containing only occasional, ambiguous, short tubular elements. We also found that neurites would extend on ECM in the presence of the microtubule depolymerizing drug, nocodazole. At 0.1 micrograms/ml nocodazole, cells on ECM produce neurites that contain a normal density of MTs. This is in contrast to the lack of neurite outgrowth and retraction of extant neurites that this dose produces in cells grown on polylysine. At 0.2 microgram/ml nocodazole, neurites again grew out in substantial number and four of five neurites examined ultrastructurally were found to be completely devoid of microtubules. We interpret these

  14. Design of 3D engineered protein hydrogels for tailored control of neurite growth

    PubMed Central

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

    2013-01-01

    The design of bioactive materials allows for tailored studies probing cell-biomaterial interactions; however, relatively few studies have examined effects of ligand density and material stiffness on neurite growth in 3D. Elastin-like proteins (ELPs) have been designed with modular bioactive and structural regions to enable the systematic characterization of design parameters within 3D materials. To promote neurite outgrowth and better understand the effects of common biomaterial design parameters on neuronal cultures, we here focused on cell-adhesive ligand density and hydrogel stiffness as design variables for ELP hydrogels. With the inherent design freedom of engineered proteins, these 3D ELP hydrogels enabled decoupled investigation into the effects of biomechanics and biochemistry on neurite outgrowth from dorsal root ganglia (DRG). Increasing the cell-adhesive RGD ligand density from 0 to 1.9 × 107 ligands/μm3 led to a significant increase in the rate, length, and density of neurite outgrowth, as quantified by a high-throughput algorithm developed for dense neurite analysis. An approximately two-fold improvement in total neurite outgrowth was observed in materials with the higher ligand density at all time-points through 7 days. ELP hydrogels with initial elastic moduli of 0.5, 1.5, or 2.1 kPa and identical RGD ligand densities revealed that the most compliant materials led to the greatest outgrowth, with some neurites extending over 1800 μm by day 7. Given the ability of ELP hydrogels to efficiently promote neurite outgrowth within defined and tunable 3D microenvironments, these materials may be useful in developing therapeutic nerve guides and the further study of basic neuron-biomaterial interactions. PMID:23128159

  15. Neuronal Survival, Morphology and Outgrowth of Spiral Ganglion Neurons Using a Defined Growth Factor Combination

    PubMed Central

    Schwieger, Jana; Warnecke, Athanasia; Lenarz, Thomas; Esser, Karl-Heinz; Scheper, Verena

    2015-01-01

    Objectives The functionality of cochlear implants (CI) depends, among others, on the number and excitability of surviving spiral ganglion neurons (SGN). The spatial separation between the SGN, located in the bony axis of the inner ear, and the CI, which is inserted in the scala tympani, results in suboptimal performance of CI patients and may be decreased by attracting the SGN neurites towards the electrode contacts. Neurotrophic factors (NTFs) can support neuronal survival and neurite outgrowth. Methods Since brain-derived neurotrophic factor (BDNF) is well known for its neuroprotective effect and ciliary neurotrophic factor (CNTF) increases neurite outgrowth, we evaluated if the combination of BDNF and CNTF leads to an enhanced neuronal survival with extended neurite outgrowth. Both NTFs were added in effective high concentrations (BDNF 50ng/ml, CNTF 100ng/ml), alone and in combination, to cultured dissociated SGN of neonatal rats for 48 hours. Results The neuronal survival and neurite outgrowth were significantly higher in SGN treated with the combination of the two NTFs compared to treatment with each factor alone. Additionally, with respect to the morphology, the combination of BDNF and CNTF leads to a significantly higher number of bipolar neurons and a decreased number of neurons without neurites in culture. Conclusion The combination of BDNF and CNTF shows a great potential to increase the neuronal survival and the number of bipolar neurons in vitro and to regenerate retracted nerve fibers. PMID:26263175

  16. Neuronal Survival, Morphology and Outgrowth of Spiral Ganglion Neurons Using a Defined Growth Factor Combination.

    PubMed

    Schwieger, Jana; Warnecke, Athanasia; Lenarz, Thomas; Esser, Karl-Heinz; Scheper, Verena

    2015-01-01

    The functionality of cochlear implants (CI) depends, among others, on the number and excitability of surviving spiral ganglion neurons (SGN). The spatial separation between the SGN, located in the bony axis of the inner ear, and the CI, which is inserted in the scala tympani, results in suboptimal performance of CI patients and may be decreased by attracting the SGN neurites towards the electrode contacts. Neurotrophic factors (NTFs) can support neuronal survival and neurite outgrowth. Since brain-derived neurotrophic factor (BDNF) is well known for its neuroprotective effect and ciliary neurotrophic factor (CNTF) increases neurite outgrowth, we evaluated if the combination of BDNF and CNTF leads to an enhanced neuronal survival with extended neurite outgrowth. Both NTFs were added in effective high concentrations (BDNF 50 ng/ml, CNTF 100 ng/ml), alone and in combination, to cultured dissociated SGN of neonatal rats for 48 hours. The neuronal survival and neurite outgrowth were significantly higher in SGN treated with the combination of the two NTFs compared to treatment with each factor alone. Additionally, with respect to the morphology, the combination of BDNF and CNTF leads to a significantly higher number of bipolar neurons and a decreased number of neurons without neurites in culture. The combination of BDNF and CNTF shows a great potential to increase the neuronal survival and the number of bipolar neurons in vitro and to regenerate retracted nerve fibers.

  17. Laminin promotes neuritic regeneration from cultured peripheral and central neurons

    PubMed Central

    1983-01-01

    The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co- purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin. PMID:6643580

  18. 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

  19. Cholinergic neuronotrophic factors: V. Segregation of survival- and neurite-promoting activities in heart-conditioned media.

    PubMed

    Adler, R; Varon, S

    1980-04-28

    Chick embryo ciliary ganglionic (CG) neurons will not survive in monolayer culture unless special supplements are provided in the medium. We have previously reported that two such supplements, chick embryo extract and medium conditioned over chick heart cell cultures (HCM) share the capacity to support survival of CG neurons but differ in their neurite-promoting effects. Thus, embryo extract elicited neuritic outgrowth only on collagen and HCM did so only on polyornithine (PORN), although both agents supported neuronal survival on both substrata. We report here the separation and quantitation of two different HCM components. One is a trophic agent which supports survival of CG neurons on either collagen or PORN, but does not seem to adsorb to either substratum. The other is a neurite-promoting factor (NPF) which adsorbs to PORN but not to collagen. Overnight incubation of HCM on PORN yields two products: (i) an NPF-deprived HCM, that has no neurite-promoting activity and (ii) an NPF-coated PORN, that promotes neuritic development of CG neurons trophically supported by either embryo extract or NPF-deprived HCM. CG requirements for neuritic outgrowth were also examined in explant cultures. No neurites were present after 24 h when explants were cultured in plain medium on PORN. Very extensive radial neuritic outgrowth was observed when explants were cultured in HCM on fresh PORN, or in NPF-deprived HCM on NPF-derivatized PORN. In contrast to what happens with dissociated cells, neuritic outgrowth was also present when ganglia were cultured in NPF-deprived HCM on fresh PORN. However, neurites grew radially only to a limited extent, after which they adopted a circular pattern grossly concentric to the ganglionic explant. It is proposed that explanted ciliary ganglia produce a neurite-promoting factor that coats the PORN substratum in widening circles.

  20. Neurites from PC12 cells are connected to each other by synapse-like structures.

    PubMed

    Jeon, Chan-Young; Jin, Jae-Kwang; Koh, Young-Ho; Chun, Wook; Choi, Ihn-Geun; Kown, Hyung-Joo; Kim, Yong-Sun; Park, Jae-Bong

    2010-10-01

    PC12 cells have been used as a model of sympathetic neurons. Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and cAMP induce neurite outgrowth from PC12 cells. cAMP induced a greater number of neurites than did NGF. In particular, we attempted to elucidate whether PC12 cell neurites, induced by several factors including NGF, bFGF, and cAMP, form synapses, and whether each neurite has presynaptic and postsynaptic properties. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), we observed that neurites are connected to each other. The connected regions presented dense core vesicles and a clathrin-coated membrane invagination. In addition, typical maker proteins for axon and dendrite were identified by an immuno-staining method. Tau-1, an axonal marker in neurons, was localized at a high concentration in the terminal tips of neurites from PC12 cells, which were connected to neurite processes containing MAP-2, a dendritic marker in neurons. Furthermore, neurites containing SV2 and synaptotagmin, markers of synaptic vesicles, were in contact with neurites harboring drebrin, a marker of the postsynaptic membrane, suggesting that neurites from PC12 cells induced by NGF, bFGF, and cAMP may form synapse-like structures. Tat-C3 toxin, a Rho inhibitor, augmented neurite outgrowth induced by NGF, bFGF, and cAMP. Tat-C3 toxin together with neurotrophins also exhibited synapse-like structures between neurites. However, it remains to be studied whether RhoA inhibition plays a role in the formation of synapse-like structures in PC12 cells. (c) 2010 Wiley-Liss, Inc.

  1. Quantitative Assessment of Neurite Outgrowth in PC12 Cells

    EPA Science Inventory

    In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity. In order to identify potential developmental neurotoxicants, assessment of critical neurodevelopmental processes such as neuronal differenti...

  2. Quantitative Assessment of Neurite Outgrowth in PC12 Cells

    EPA Science Inventory

    In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity. In order to identify potential developmental neurotoxicants, assessment of critical neurodevelopmental processes such as neuronal differenti...

  3. A disintegrin and metalloproteinase 12 (ADAM12) localizes to invasive trophoblast, promotes cell invasion and directs column outgrowth in early placental development.

    PubMed

    Aghababaei, M; Perdu, S; Irvine, K; Beristain, A G

    2014-03-01

    During pregnancy, stromal- and vascular-remodeling trophoblasts serve critical roles in directing placental development acquiring pro-invasive characteristics. The A Disintegrin and Metalloproteinase (ADAM) family of multifunctional proteins direct cellular processes across multiple organ systems via their intrinsic catalytic, cell adhesive and intracellular signaling properties. ADAM12, existing as two distinct splice variants (ADAM12L and ADAM12S), is highly expressed in the human placenta and promotes cell migration and invasion in several tumor cell lines; however, its role in trophoblast biology is unknown. In this study, ADAM12 was localized to anchoring trophoblast columns in first trimester placentas and to highly invasive extracellular matrix-degrading trophoblasts in placental villous explants. The importance of ADAM12 in directing trophoblast invasion was tested using loss-of and gain-of-function strategies, where siRNA-directed knockdown of ADAM12 inhibited trophoblast cell invasion while over-expression promoted migration and invasion in two trophoblastic cell models. In placental villous explant cultures, siRNA-directed loss of ADAM12 significantly dampened trophoblast column outgrowth. Additionally, we provide functional evidence for the ADAM12S variant in promoting trophoblast invasion and column outgrowth through a mechanism requiring its catalytic activity. This is the first study to assign a function for ADAM12 in trophoblast biology, where ADAM12 may play a central role regulating the behavior of invasive trophoblast subsets in early pregnancy. This study also underlines the importance of ADAM12L and ADAM12S in directing cell motility in normal developmental processes outside of cancer, specifically highlighting a potentially important function of ADAM12S in directing early placental development.

  4. Robust Neurite Extension Following Exogenous Electrical Stimulation within Single Walled Carbon Nanotube-Composite Hydrogels

    PubMed Central

    Koppes, A. N.; Keating, K. W.; McGregor, A. L.; Koppes, R. A.; Kearns, K. R.; Ziemba, A. M.; McKay, C. A.; Zuidema, J. M.; Rivet, C. J.; Gilbert, R. J.; Thompson, D. M.

    2016-01-01

    The use of exogenous electrical stimulation to promote nerve regeneration has achieved only limited success. Conditions impeding optimized outgrowth may arise from inadequate stimulus presentation due to differences in injury geometry or signal attenuation. Implantation of an electrically-conductive biomaterial may mitigate this attenuation and provide a more reproducible signal. In this study, a conductive nanofiller (single-walled carbon nanotubes [SWCNT]) was selected as one possible material to manipulate the bulk electrical properties of a collagen type I-10% Matrigel™ composite hydrogel. Neurite outgrowth within hydrogels (SWCNT or nanofiller-free controls) was characterized to determine if: 1) nanofillers influence neurite extension and 2) electrical stimulation of the nanofiller composite hydrogel enhances neurite outgrowth. Increased SWCNT loading (10–100-μg/ml) resulted in greater bulk conductivity (up to 1.7-fold) with no significant changes to elastic modulus. Neurite outgrowth increased 3.3-fold in 20-μg/mL SWCNT loaded biomaterials relative to the nanofiller-free control. Electrical stimulation promoted greater outgrowth (2.9-fold) within SWCNT-free control. The concurrent presentation of electrical stimulation and SWCNT-loaded biomaterials resulted in a 7.0-fold increase in outgrowth relative to the unstimulated, nanofiller-free controls. Local glia residing within the DRG likely contribute, in part, to the observed increases in outgrowth; but it is unknown which specific nanofiller properties influence neurite extension. Characterization of neuronal behavior in model systems, such as those described here, will aid the rational development of biomaterials as well as the appropriate delivery of electrical stimuli to support nerve repair. PMID:27167609

  5. Androgen regulation of axon growth and neurite extension in motoneurons

    PubMed Central

    Fargo, Keith N.; Galbiati, Mariarita; Foecking, Eileen M.; Poletti, Angelo; Jones, Kathryn J.

    2008-01-01

    Androgens act on the CNS to affect motor function through interaction with a widespread distribution of intracellular androgen receptors (AR). This review highlights our work on androgens and process outgrowth in motoneurons, both in vitro and in vivo. The actions of androgens on motoneurons involve the generation of novel neuronal interactions that are mediated by the induction of androgen-dependent neurite or axonal outgrowth. Here, we summarize the experimental evidence for the androgenic regulation of the extension and regeneration of motoneuron neurites in vitro using cultured immortalized motoneurons, and axons in vivo using the hamster facial nerve crush paradigm. We place particular emphasis on the relevance of these effects to SBMA and peripheral nerve injuries. PMID:18387610

  6. Sigma-1 receptor enhances neurite elongation of cerebellar granule neurons via TrkB signaling.

    PubMed

    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 Ca(2+) 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.

  7. 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

  8. Positive and negative cues for modulating neurite dynamics and receptor expression.

    PubMed

    Wrobel, Melissa R; Sundararaghavan, Harini G

    2017-03-27

    Many current peripheral nerve repair strategies focus on delivering positive, growth promoting cues (e.g. extracellular matrix, ECM) while eliminating negative, growth inhibiting cues (e.g. chondroitin sulfate proteoglycans, CSPGs) at the injury site. We hypothesized that recapitulating the positive and negative cues of the peripheral nerve injury microenvironment would improve regeneration. First, we tested the effects of a characteristic CSPG, chondroitin sulfate A (CSA) on neurite dynamics of dissociated chick embryo dorsal root ganglion (DRG) neurons using time lapse video microscopy. DRG growth was recorded on different adhesive substrates, including a novel, porcine-derived spinal cord matrix (SCM). The SCM significantly increased frequency of neurite extension coordinated by a significant reduction in the neurites' time spent stalled. The SCM also mitigated inhibitory effects of CSA, producing longer neurites than the controls without CSA treatment. Next we aimed to elucidate receptors involved in mediating this behavior by testing the ability of CSA to upregulate cell-substrate binding receptors using flow cytometry. Our results showed a significant increase in syndecan-3 receptor expression in neurons treated with CSA. Furthermore, syndecans would most likely bind to the sulfated glycosaminoglycans measured in the SCM. Finally, we evaluated neurite growth on biomaterial scaffolds featuring CSA and SCM cues. Our results showed significantly increased neurite outgrowth on electrospun hyaluronic acid fibers with SCM and low levels of CSA. Higher incorporation of CSA maintained its inhibitory properties. Future work will evaluate coupling CSPGs with growth-permissive ECM to assess the combined effect on neurite outgrowth.

  9. Early-outgrowth bone marrow cells attenuate renal injury and dysfunction via an antioxidant effect in a mouse model of type 2 diabetes.

    PubMed

    Zhang, Yanling; Yuen, Darren A; Advani, Andrew; Thai, Kerri; Advani, Suzanne L; Kepecs, David; Kabir, M Golam; Connelly, Kim A; Gilbert, Richard E

    2012-08-01

    Cell therapy has been extensively investigated in heart disease but less so in the kidney. We considered whether cell therapy also might be useful in diabetic kidney disease. Cognizant of the likely need for autologous cell therapy in humans, we sought to assess the efficacy of donor cells derived from both healthy and diabetic animals. Eight-week-old db/db mice were randomized to receive a single intravenous injection of PBS or 0.5 × 10(6) early-outgrowth cells (EOCs) from db/m or db/db mice. Effects were assessed 4 weeks after cell infusion. Untreated db/db mice developed mesangial matrix expansion and tubular epithelial cell apoptosis in association with increased reactive oxygen species (ROS) and overexpression of thioredoxin interacting protein (TxnIP). Without affecting blood glucose or blood pressure, EOCs not only attenuated mesangial and peritubular matrix expansion, as well as tubular apoptosis, but also diminished ROS and TxnIP overexpression in the kidney of db/db mice. EOCs derived from both diabetic db/db and nondiabetic db/m mice were equally effective in ameliorating kidney injury and oxidative stress. The similarly beneficial effects of cells from healthy and diabetic donors highlight the potential of autologous cell therapy in the related clinical setting.

  10. Effects of DDT and permethrin on neurite growth in cultured neurons of chick embryo brain and Lymnaea stagnalis.

    PubMed

    Ferguson, C A; Audesirk, G

    1990-01-01

    The pesticides permethrin and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT), dissolved in either ethanol (EtOH) or dimethylsulphoxide (DMSO), were studied to determine their effect on neurite growth from cultured neurons of Lymnaea stagnalis and embryonic chicks. Both of these toxins decreased the percentage of neurons growing neurites, mean neurite length, and number of neurites/cell in a dose-dependent manner. DMSO increased the toxicity of permethrin and DDT in L. stagnalis neurons. EtOH was not used as a solvent with the embryonic chick cultures. Pre-existing neurites of L. stagnalis neurons exposed to permethrin regressed in a dose- and time-dependent manner. These two toxins may affect neurite outgrowth through interference with intracellular calcium regulation.

  11. 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.

  12. Effects of surface charges of graphene oxide on neuronal outgrowth and branching.

    PubMed

    Tu, Qin; Pang, Long; Chen, Yun; Zhang, Yanrong; Zhang, Rui; Lu, Bingzhang; Wang, Jinyi

    2014-01-07

    Graphene oxides with different surface charges were fabricated from carboxylated graphene oxide by chemical modification with amino- (-NH2), poly-m-aminobenzene sulfonic acid- (-NH2/-SO3H), or methoxyl- (-OCH3) terminated functional groups. The chemically functionalized graphene oxides and the carboxylated graphene oxide were characterized by infrared spectroscopy, X-ray photoelectron spectroscopy, UV-Vis spectrometry, ζ potential measurements, field emission scanning electron microscopy, and contact angle analyses. Subsequently, the resulting graphene oxides were used as substrates for culturing primary rat hippocampal neurons to investigate neurite outgrowth and branching. The morphological features of neurons that directly reflect their potential capability in synaptic transmission were characterized. The results demonstrate that the chemical properties of graphene oxide can be systematically modified by attaching different functional groups that confer known characteristics to the substrate. By manipulating the charge carried by the functionalized graphene oxides, the outgrowth and branching of neuronal processes can be controlled. Compared with neutral, zwitterionic, or negatively charged graphene oxides, positively charged graphene oxide was found to be more beneficial for neurite outgrowth and branching. The ability to chemically modify graphene oxide to control neurite outgrowth could be implemented clinically, especially in cases wherein long-term presence of outgrowth modulation is necessary.

  13. 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.

  14. GEFs and Rac GTPases control directional specificity of neurite extension along the anterior–posterior axis

    PubMed Central

    Zheng, Chaogu; Diaz-Cuadros, Margarete; Chalfie, Martin

    2016-01-01

    Although previous studies have identified many extracellular guidance molecules and intracellular signaling proteins that regulate axonal outgrowth and extension, most were conducted in the context of unidirectional neurite growth, in which the guidance cues either attract or repel growth cones. Very few studies addressed how intracellular signaling molecules differentially specify bidirectional outgrowth. Here, using the bipolar PLM neurons in Caenorhabditis elegans, we show that the guanine nucleotide exchange factors (GEFs) UNC-73/Trio and TIAM-1 promote anterior and posterior neurite extension, respectively. The Rac subfamily GTPases act downstream of the GEFs; CED-10/Rac1 is activated by TIAM-1, whereas CED-10 and MIG-2/RhoG act redundantly downstream of UNC-73. Moreover, these two pathways antagonize each other and thus regulate the directional bias of neuritogenesis. Our study suggests that directional specificity of neurite extension is conferred through the intracellular activation of distinct GEFs and Rac GTPases. PMID:27274054

  15. 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.

  16. Quantifying Spiral Ganglion Neurite and Schwann Behavior on Micropatterned polymer Substrates

    PubMed Central

    Cheng, Elise L.; Leigh, Braden; Guymon, C. Allan; Hansen, Marlan R.

    2017-01-01

    The first successful in vitro experiments on the cochlea were conducted in 1928 by Honor Fell [1]. 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 has been utilized in recent years [2,3]. 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

  17. A retinoic acid receptor β agonist (CD2019) overcomes inhibition of axonal outgrowth via phosphoinositide 3-kinase signalling in the injured adult spinal cord

    PubMed Central

    Agudo, Marta; Yip, Ping; Davies, Meirion; Bradbury, Elizabeth; Doherty, Patrick; McMahon, Stephen; Maden, Malcolm; Corcoran, Jonathan P.T.

    2010-01-01

    After spinal cord injury in the adult mammal, axons do not normally regrow and this commonly leads to paralysis. Retinoic acid (RA) can stimulate neurite outgrowth in vitro of both the embryonic central and peripheral nervous system, via activation of the retinoic acid receptor (RAR) β2. We show here that regions of the adult CNS, including the cerebellum and cerebral cortex, express RARβ2. We show that when cerebellar neurons are grown in the presence of myelin-associated glycoprotein (MAG) which inhibits neurite outgrowth, RARβ can be activated in a dose dependent manner by a RARβ agonist (CD2019) and neurite outgrowth can occur via phosphoinositide 3-kinase (PI3K) signalling. In a model of spinal cord injury CD2019 also acts through PI3K signalling to induce axonal outgrowth of descending corticospinal fibres and promote functional recovery. Our data suggest that RARβ agonists may be of therapeutic potential for human spinal cord injuries. PMID:19800972

  18. A retinoic acid receptor beta agonist (CD2019) overcomes inhibition of axonal outgrowth via phosphoinositide 3-kinase signalling in the injured adult spinal cord.

    PubMed

    Agudo, Marta; Yip, Ping; Davies, Meirion; Bradbury, Elizabeth; Doherty, Patrick; McMahon, Stephen; Maden, Malcolm; Corcoran, Jonathan P T

    2010-01-01

    After spinal cord injury in the adult mammal, axons do not normally regrow and this commonly leads to paralysis. Retinoic acid (RA) can stimulate neurite outgrowth in vitro of both the embryonic central and peripheral nervous system, via activation of the retinoic acid receptor (RAR) beta2. We show here that regions of the adult CNS, including the cerebellum and cerebral cortex, express RARbeta2. We show that when cerebellar neurons are grown in the presence of myelin-associated glycoprotein (MAG) which inhibits neurite outgrowth, RARbeta can be activated in a dose dependent manner by a RARbeta agonist (CD2019) and neurite outgrowth can occur via phosphoinositide 3-kinase (PI3K) signalling. In a model of spinal cord injury CD2019 also acts through PI3K signalling to induce axonal outgrowth of descending corticospinal fibres and promote functional recovery. Our data suggest that RARbeta agonists may be of therapeutic potential for human spinal cord injuries.

  19. Spiral ganglion outgrowth and hearing development in p75-deficient mice.

    PubMed

    Brors, Dominik; Hansen, Stefan; Mlynski, Robert; Volkenstein, Stefan; Aletsee, Christoph; Sendtner, Michael; Ryan, Allen F; Dazert, Stefan

    2008-01-01

    To explore the role of nerve growth factor receptor p75(NTR) during the terminal neuronal development of the mammalian cochlea the onset of hearing and the in vitro response of spiral ganglion neurites to neurotrophin 3 (NT-3), which is known to play a critical role during neonatal inner ear development, were investigated in p75(NTR)-deficient mice (p75(NTR)-/-). Auditory-evoked brain stem response recordings from p75(NTR)-/- and wild-type (WT) littermates were measured from postnatal days (PD) 8 to 23. Additionally, spiral ganglion explants from p75(NTR)-/- and WT animals were dissected and cultured in an organotypic tissue culture system. In both groups, spiral ganglion neurite outgrowth was analyzed with and without NT-3 supplementation. No significant differences in the onset of hearing of mutant mice compared to the WT mice were detected, and both groups showed a similar development of hearing until PD 23. After stimulation with NT-3, neurite outgrowth was enhanced in both p75(NTR)-/- and WT mice. However, neurites from p75(NTR)-/- spiral ganglion explants were longer in both culture conditions. Moreover, NT-3 did not significantly enhance neurite number in p75(NTR)-/-, as it did in WT mice. P75(NTR) has a remarkable influence on spiral ganglion neurite growth behavior. However, p75(NTR) does not seem to be essential for the development of basic hearing function in the first 3 postnatal weeks. Copyright 2008 S. Karger AG, Basel.

  20. 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