Science.gov

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. 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.

  3. 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.

  4. 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

  5. Optimizing neurotrophic factor combinations for neurite outgrowth

    NASA Astrophysics Data System (ADS)

    Deister, C.; Schmidt, C. E.

    2006-06-01

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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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).

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. ANALYSIS OF THE STRUCTURE OF MAGNETIC FIELDS THAT INDUCED INHIBITION OF STIMULATED NEURITE OUTGROWTH

    EPA Science Inventory

    The important experiments showing nonlinear amplitude dependences of the neurite outgrowth in pheochromocytoma nerve cells due to ELF magnetic field exposure had been carried out in a nonuniform ac magnetic field. The nonuniformity entailed larger than expected variances in magne...

  7. 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

  8. 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.

  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. 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

  11. 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.

  12. 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

  13. Analysis of the action of euxanthone, a plant-derived compound that stimulates neurite outgrowth.

    PubMed

    Naidu, M; Kuan, C-Y K; Lo, W-L; Raza, M; Tolkovsky, A; Mak, N-K; Wong, R N-S; Keynes, R

    2007-09-21

    We have investigated the neurite growth-stimulating properties of euxanthone, a xanthone derivative isolated from the Chinese medicinal plant Polygala caudata. Euxanthone was shown to exert a marked stimulatory action on neurite outgrowth from chick embryo dorsal root ganglia explanted in collagen gels, in the absence of added neurotrophins. It was also shown to promote cell survival in explanted chick embryo ganglia, and to stimulate neurite outgrowth from isolated adult rat primary sensory neurons in vitro. The further finding that euxanthone stimulates neurite outgrowth from explants of chick embryo retina and ventral spinal cord suggests an action on signaling pathways downstream of neuronal receptors for specific neurotrophic factors. Consistent with this, euxanthone did not promote neurite outgrowth from non-transfected PC12 cells, or from PC12 cells transfected with TrkB or TrkC, under conditions in which these cells extended neurites in response to, respectively, the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3. Western blot analysis of euxanthone-stimulated dorsal root ganglion explants showed that expression of phospho-mitogen-activated protein (MAP) kinase was up-regulated after 1 h of euxanthone-treatment. Inhibition of the MAP kinase pathway using PD98059, a specific inhibitor of MAP kinase kinase, blocked all euxanthone-stimulated neurite outgrowth. However, analysis of phospho-Akt expression indicated that the phosphatidylinositol-3 kinase-Akt pathway, another major signaling pathway engaged by neurotrophins, is not significantly activated by euxanthone. These results suggest that euxanthone promotes neurite outgrowth by selectively activating the MAP kinase pathway.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  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. 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

  3. 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

  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. 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

  7. 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.

  8. 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...

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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).

  17. 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

  18. 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...

  19. 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

  20. Neurite outgrowth-promoting active constituents of the Japanese cypress (Chamaecyparis obtusa).

    PubMed

    Kuroyanagi, Masanori; Ikeda, Ryuya; Gao, Hui Yuan; Muto, Norio; Otaki, Keisuke; Sano, Toshikazu; Kawahara, Nobuo; Nakane, Takahisa

    2008-01-01

    In the screening of biologically active constituents from woody plants, the methanol extract of leaves of Chamaecyparis obtusa showed potent neurite outgrowth-promoting activity in neuronal PC12 cells. The ethyl acetate-soluble fraction of the methanol extract showed potent activity and was separated by means of various chromatographic methods to give the two new compounds 1 and 2, as well as 11 known lignan and sesquiterpene derivatives. The structures of the new compounds were determined to be 9-O-acetyldihydrosesamin (1) and 9-O-(11-hydroxyeudesman-4-yl)dihydrosesamin (2), respectively, in NMR studies including 2D-NMR experiments. Of the 13 compounds, the known compound hinokinin (5) and the new compound 2 showed potent neurite outgrowth-promoting activity in PC 12 cells.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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...

  18. 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...

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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...

  16. 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

  17. 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.

  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. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. CaMKII-Mediated CREB Phosphorylation Is Involved in Ca2+-Induced BDNF mRNA Transcription and Neurite Outgrowth Promoted by Electrical Stimulation

    PubMed Central

    Ye, Zhengxu; Huang, Jinghui; He, Fei; Xiao, Wei; Hu, Xueyu; Luo, Zhuojing

    2016-01-01

    Electrical stimulation (ES)-triggered up-regulation of brain-derived neurotrophic factor (BDNF) and neurite outgrowth in cultured rat postnatal dorsal root ganglion neurons (DRGNs) is calcium (Ca2+)-dependent. The effects of increased Ca2+ on BDNF up-regulation and neurite outgrowth remain unclear. We showed here that ES increased phosphorylation of the cAMP-response element binding protein (CREB). Blockade of Ca2+ suppressed CREB phosphorylation and neurite outgrowth. Down-regulation of phosphorylated (p)-CREB reduced BDNF transcription and neurite outgrowth triggered by ES. Furthermore, blockade of calmodulin-dependent protein kinase II (CaMKII) using the inhibitors KN93 or KN62 reduced p-CREB, and specific knockdown of the CaMKIIα or CaMKIIβ subunit was sufficient to suppress p-CREB. Recombinant BDNF or hyperforin reversed the effects of Ca2+ blockade and CaMKII knockdown. Taken together, these data establish a potential signaling pathway of Ca2+-CaMKII-CREB in neuronal activation. To our knowledge, this is the first report of the mechanisms of Ca2+-dependent BDNF transcription and neurite outgrowth triggered by ES. These findings might help further investigation of complex molecular signaling networks in ES-triggered nerve regeneration in vivo. PMID:27611779

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  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. 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.

  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. 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.

  5. 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.

  6. 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.

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

    PubMed Central

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

    2012-01-01

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

  8. A 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

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

    PubMed

    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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

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

    PubMed Central

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

    2015-01-01

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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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).

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  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. 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.

  13. 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.

  14. 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

  15. 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).

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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...

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  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. Pure neuritic leprosy: Current status and relevance.

    PubMed

    Rao, P Narasimha; Suneetha, Sujai

    2016-01-01

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

  20. Multifunctional Silk Nerve Guides for Axon Outgrowth

    NASA Astrophysics Data System (ADS)

    Tupaj, Marie C.

    Peripheral nerve regeneration is a critical issue as 2.8% of trauma patients present with this type of injury, estimating a total of 200,000 nerve repair procedures yearly in the United States. While the peripheral nervous system exhibits slow regeneration, at a rate of 0.5 mm -- 9 mm/day following trauma, this regenerative ability is only possible under certain conditions. Clinical repairs have changed slightly in the last 30 years and standard methods of treatment include suturing damaged nerve ends, allografting, and autografting, with the autograft the gold standard of these approaches. Unfortunately, the use of autografts requires a second surgery and there is a shortage of nerves available for grafting. Allografts are a second option however allografts have lower success rates and are accompanied by the need of immunosuppressant drugs. Recently there has been a focus on developing nerve guides as an "off the shelf" approach. Although some natural and synthetic guidance channels have been approved by the FDA, these nerve guides are unfunctionalized and repair only short gaps, less than 3 cm in length. The goal of this project was to identify strategies for functionalizing peripheral nerve conduits for the outgrowth of neuron axons in vitro . To accomplish this, two strategies (bioelectrical and biophysical) were indentified for increasing axon outgrowth and promoting axon guidance. Bioelectrical strategies exploited electrical stimulation for increasing neurite outgrowth. Biophysical strategies tested a range of surface topographies for axon guidance. Novel methods were developed for integrating electrical and biophysical strategies into silk films in 2D. Finally, a functionalized nerve conduit system was developed that integrated all strategies for the purpose of attaching, elongating, and guiding nervous tissue in vitro. Future directions of this work include silk conduit translation into a rat sciatic nerve model in vivo for the purpose of repairing long

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

    PubMed Central

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

    2016-01-01

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

  2. Effects of Oleanane-Type Triterpene Saponins from the Leaves of Eleutherococcus senticosus in an Axonal Outgrowth Assay.

    PubMed

    Ge, Yue-Wei; Tohda, Chihiro; Zhu, Shu; He, Yu-Min; Yoshimatsu, Kayo; Komatsu, Katsuko

    2016-07-22

    An aqueous extract of Eleutherococcus senticosus leaves exerted a beneficial effect in restoring the neurite outgrowth from Aβ25-35-induced degeneration using an axonal density assay. Subsequent bioassay-guided fractionation afforded seven new oleanane-type triterpene saponins, ezoukoginosides A-G (1-7), along with nine known analogues. The structures of 1-7 were elucidated through chemical and spectroscopic approaches, and their effects on restoring the neurite outgrowth from Aβ25-35-induced degeneration were investigated. The results revealed that hydrophilic oleanane-type saponins substituted with a free carboxylic acid, hydroxy, or formyl group in the aglycone, especially when the oxidation occurred at C-29, not only restrained Aβ25-35-induced degeneration but also restored axonal outgrowth significantly. Compounds 2 (-COOH at C-29) and 3 (-CH2OH at C-29) showed the most potent bioactivity among the isolates.

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

    PubMed

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

    2016-04-20

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

  4. Tgfβ2 and 3 are coexpressed with their extracellular regulator Ltbp1 in the early limb bud and modulate mesodermal outgrowth and BMP signaling in chicken embryos

    PubMed Central

    2010-01-01

    Background Transforming growth factor β proteins (Tgfβs) are secreted cytokines with well-defined functions in the differentiation of the musculoskeletal system of the developing limb. Here we have studied in chicken embryos, whether these cytokines are implicated in the development of the embryonic limb bud at stages preceding tissue differentiation. Results Immunohistochemical detection of phosphorylated Smad2 and Smad3 indicates that signaling by this pathway is active in the undifferentiated mesoderm and AER. Gene expression analysis shows that transcripts of tgfβ2 and tgfβ3 but not tgfβ1 are abundant in the growing undifferentiated limb mesoderm. Transcripts of tgfβ2 are also found in the AER, which is the signaling center responsible for limb outgrowth. Furthermore, we show that Latent Tgfβ Binding protein 1 (LTBP1), which is a key extracellular modulator of Tgfβ ligand bioavailability, is coexpressed with Tgfβs in the early limb bud. Administration of exogenous Tgfβs to limb buds growing in explant cultures provides evidence of these cytokines playing a role in the regulation of mesodermal limb proliferation. In addition, analysis of gene regulation in these experiments revealed that Tgfβ signaling has no effect on the expression of master genes of musculoskeletal tissue differentiation but negatively regulates the expression of the BMP-antagonist Gremlin. Conclusion We propose the occurrence of an interplay between Tgfβ and BMP signaling functionally associated with the regulation of early limb outgrowth by modulating limb mesenchymal cell proliferation. PMID:20565961

  5. Nitric oxide acts as a slow-down and search signal in developing neurites.

    PubMed

    Trimm, Kevin R; Rehder, Vincent

    2004-02-01

    Nitric oxide (NO) has been demonstrated to act as a signaling molecule during neuronal development, but its precise function is unclear. Here we investigate whether NO might function at the neuronal growth cone to affect growth cone motility. We have previously demonstrated that growth cones of identified neurons from the snail Helisoma trivolvis show a rapid and transient increase in filopodial length in response to NO, which was regulated by soluble guanylyl cyclase (sGC) [S. Van Wagenen and V. Rehder (1999) J. Neurobiol., 39, 168-185]. Because in vivo studies have demonstrated that growth cones have longer filopodia and advance more slowly in regions where pathfinding decisions are being made, this study aimed to establish whether NO could function as a combined 'slow-down and search signal' for growth cones by decreasing neurite outgrowth. In the presence of the NO donor NOC-7, neurites of B5 neurons showed a concentration-dependent effect on neurite outgrowth, ranging from slowing at low, stopping at intermediate and collapsing at high concentrations. The effects of the NO donor were mimicked by directly activating sGC with YC-1, or by increasing its product with 8-bromo-cGMP. In addition, blocking sGC in the presence of NO with NS2028 blocked the effect of NO, suggesting that NO affected outgrowth via sGC. Ca2+ imaging of growth cones with Fura-2 indicated that [Ca2+]i increased transiently in the presence of NOC-7. These results support the hypothesis that NO can function as a potent slow/stop signal for developing neurites. When coupled with transient filopodia elongation, this phenomenon emulates growth cone searching behavior.

  6. Mechanisms of developmental neurite pruning

    PubMed Central

    Schuldiner, Oren; Yaron, Avraham

    2016-01-01

    The precise wiring of the nervous system is a combined outcome of progressive and regressive events during development. Axon guidance and synapse formation intertwined with cell death and neurite pruning sculpt the mature circuitry. It is now well recognized that pruning of dendrites and axons as means to refine neuronal networks, is a wide spread phenomena required for the normal development of vertebrate and invertebrate nervous systems. Here we will review the arising principles of cellular and molecular mechanisms of neurite pruning. We will discuss these principles in light of studies in multiple neuronal systems, and speculate on potential explanations for the emergence of neurite pruning as a mechanism to sculpt the nervous system. PMID:25213356

  7. Molecular mechanisms of neurite extension.

    PubMed Central

    Valtorta, F; Leoni, C

    1999-01-01

    The extension of neurites is a major task of developing neurons, requiring a significant metabolic effort to sustain the increase in molecular synthesis necessary for plasma membrane expansion. In addition, neurite extension involves changes in the subsets of expressed proteins and reorganization of the cytomatrix. These phenomena are driven by environmental cues which activate signal transduction processes as well as by the intrinsic genetic program of the cell. The present review summarizes some of the most recent progress made in the elucidation of the molecular mechanisms underlying these processes. PMID:10212488

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

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

    NASA Astrophysics Data System (ADS)

    Recho, Pierre; Jerusalem, Antoine; Goriely, Alain

    2016-03-01

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

  10. Aberrant Neuronal Differentiation and Inhibition of Dendrite Outgrowth Resulting from Endoplasmic Reticulum Stress

    PubMed Central

    Kawada, Koichi; Iekumo, Takaaki; Saito, Ryo; Kaneko, Masayuki; Mimori, Seisuke; Nomura, Yasuyuki; Okuma, Yasunobu

    2014-01-01

    Neural stem cells (NSCs) play an essential role in development of the central nervous system. Endoplasmic reticulum (ER) stress induces neuronal death. After neuronal death, neurogenesis is generally enhanced to repair the damaged regions. However, it is unclear whether ER stress directly affects neurogenesis-related processes such as neuronal differentiation and dendrite outgrowth. We evaluated whether neuronal differentiation and dendrite outgrowth were regulated by HRD1, a ubiquitin ligase that was induced under mild conditions of tunicamycin-induced ER stress. Neurons were differentiated from mouse embryonic carcinoma P19 cells by using retinoic acid. The differentiated cells were cultured for 8 days with or without tunicamycin and HRD1 knockdown. The ER stressor led to markedly increased levels of ER stress. ER stress increased the expression levels of neuronal marker βIII-tubulin in 8-day-differentiated cells. However, the neurites of dendrite marker microtubule-associated protein-2 (MAP-2)-positive cells appeared to retract in response to ER stress. Moreover, ER stress markedly reduced the dendrite length and MAP-2 expression levels, whereas it did not affect the number of surviving mature neurons. In contrast, HRD1 knockdown abolished the changes in expression of proteins such as βIII-tubulin and MAP-2. These results suggested that ER stress caused aberrant neuronal differentiation from NSCs followed by the inhibition of neurite outgrowth. These events may be mediated by increased HRD1 expression. PMID:24723324

  11. Ethanol-induced disruption of Golgi apparatus morphology, primary neurite number and cellular orientation in developing cortical neurons.

    PubMed

    Powrozek, Teresa A; Olson, Eric C

    2012-11-01

    Prenatal ethanol exposure disrupts cortical neurite initiation and outgrowth, but prior studies have reported both ethanol-dependent growth promotion and inhibition. To resolve this ambiguity and better approximate in vivo conditions, we quantitatively analyzed neuronal morphology using a new, whole hemisphere explant model. In this model, Layer 6 (L6) cortical neurons migrate, laminate and extend neurites in an organotypic fashion. To selectively label L6 neurons, we performed ex utero electroporation of a GFP expression construct at embryonic day 13 and allowed the explants to develop for 2 days in vitro. Explants were exposed to (400 mg/dL) ethanol for either 4 or 24 h prior to fixation. Complete 3-D reconstructions were made of >80 GFP-positive neurons in each experimental condition. Acute responses to ethanol exposure included compaction of the Golgi apparatus accompanied by elaboration of supernumerary primary apical neurites, as well as a modest (∼15%) increase in higher order apical neurite length. With longer exposure time, ethanol exposure leads to a consistent, significant disorientation of the cell (cell body, primary apical neurite, and Golgi) with respect to the pial surface. The effects on cellular orientation were accompanied by decreased expression of cytoskeletal elements, microtubule-associated protein 2 and F-actin. These findings indicate that upon exposure to ethanol, developing L6 neurons manifest disruptions in Golgi apparatus and cytoskeletal elements which may in turn trigger selective and significant perturbations to primary neurite formation and neuronal polarity.

  12. Microglia enhance dorsal root ganglion outgrowth in Schwann cell cultures.

    PubMed

    Hynds, Dianna L; Rangappa, Nagarathnamma; Ter Beest, Julia; Snow, Diane M; Rabchevsky, Alexander G

    2004-04-15

    Transplantation of cellular populations to facilitate regrowth of damaged axons is a common experimental therapy for spinal cord injury. Schwann cells (SC) or microglia grafted into injury sites can promote axonal regrowth of central projections of dorsal root ganglion (DRG) sensory neurons. We sought to determine whether the addition of microglia or microglia-derived secretory products alters DRG axon regrowth upon cultures of SC. Rat DRG explants were grown on monolayers consisting of either SC, microglia, SC exposed to microglia-conditioned medium (MCM), or co-cultures with different relative concentrations of microglia. Image analysis revealed that, compared to SC alone, the extent of neurite outgrowth was significantly greater on SC-microglia co-cultures. Immunocytochemistry for extracellular matrix molecules showed that microglial cells stained positively for growth-promoting thrombospondin, whereas laminin and the inhibitory chondroitin sulfate proteoglycans (CSPGs) were localized primarily to SC. Notably, immunoreactivity for CSPGs appeared reduced in areas associated with DRG outgrowth in co-cultures and SC exposed to MCM. These results show that microglia or their secreted products can augment SC-mediated DRG regrowth in vitro, indicating that co-grafting SC with microglia provides a novel approach to augment sensory fiber regeneration after spinal cord injury.

  13. GAP-43 overexpression in adult mouse Purkinje cells overrides myelin-derived inhibition of neurite growth.

    PubMed

    Gianola, Sara; Rossi, Ferdinando

    2004-02-01

    Up-regulation of growth-associated proteins in adult neurons promotes axon regeneration and neuritic elongation onto nonpermissive substrates. To investigate the interaction between these molecules and myelin-related inhibitory factors, we examined transgenic mice in which overexpression of the growth-associated protein GAP-43 is driven by the Purkinje cell-specific promoter L7. Contrary to their wild-type counterparts, which have extremely poor regenerative capabilities, axotomized transgenic Purkinje cells exhibit profuse sprouting along the intracortical neurite and at the severed stump [Buffo et al. (1997) J. Neurosci., 17, 8778-8791]. Here, we investigated the relationship between such sprouting axons and oligodendroglia to ask whether GAP-43 overexpression enables Purkinje neurites to overcome myelin-derived inhibition. Intact transgenic Purkinje axons display normal morphology and myelination. Following injury, however, many GAP-43-overexpressing neurite stumps are devoid of myelin cover and sprout into white matter regions containing densely packed myelin and Nogo-A- or MAG-immunopositive oligodendrocytes. The intracortical segments of these neurites show focal accumulations of GAP-43, which are associated with disrupted or retracted myelin sheaths. Numerous sprouts originate from such demyelinated segments and spread into the granular layer. Some myelin loss, though not axon sprouting, is also evident in wild-type mice, but this phenomenon is definitely more rapid and extensive in transgenic cerebella. Thus, GAP-43-overexpressing Purkinje axons are endowed with enhanced capabilities for growing into nonpermissive territories and show a pronounced tendency to lose myelin. Our observations suggest that accumulation of GAP-43 along precise axon segments disrupts the normal axon-glia interaction and enhances the retraction of oligodendrocytic processes to facilitate the outgrowth of neuritic sprouts.

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

    PubMed

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

    2014-05-14

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. A eudesmane-type sesquiterpene isolated from Pluchea odorata (L.) Cass. combats three hallmarks of cancer cells: Unrestricted proliferation, escape from apoptosis and early metastatic outgrowth in vitro.

    PubMed

    Blaschke, Michael; McKinnon, Ruxandra; Nguyen, Chi Huu; Holzner, Silvio; Zehl, Martin; Atanasov, Atanas Georgiev; Schelch, Karin; Krieger, Sigurd; Diaz, Rene; Frisch, Richard; Feistel, Björn; Jäger, Walter; Ecker, Gerhard F; Dirsch, Verena M; Grusch, Michael; Zupko, Istvan; Urban, Ernst; Kopp, Brigitte; Krupitza, Georg

    2015-07-01

    Pluchea odorata is ethno pharmaceutically used to treat inflammation-associated disorders. The dichloromethane extract (DME) was tested in the carrageenan-induced rat paw oedema assay investigating its effect on inflammation that was inhibited by 37%. Also an in vitro anti-neoplastic potential was reported. However, rather limited information about the bio-activity of purified compounds and their cellular mechanisms are available. Therefore, two of the most abundant eudesmanes in P. odorata were isolated and their anti-neoplastic and anti-intravasative activities were studied. HL-60 cells were treated with P. odorata compounds and metabolic activity, cell number reduction, cell cycle progression and apoptosis induction were correlated with relevant protein expression. Tumour cell intravasation through lymph endothelial monolayers was measured and potential causal mechanisms were analyzed by Western blotting. Compound PO-1 decreased the metabolic activity of HL-60 cells (IC50 = 8.9 μM after 72 h) and 10 μM PO-1 induced apoptosis, while PO-2 showed just weak anti-neoplastic activities at concentrations beyond 100 μM. PO-1 arrested the cell cycle in G1 and this correlated with induction of JunB expression. Independent of this mechanism 25 μM PO-1 decreased MCF-7 spheroid intravasation through the lymph endothelial barrier. Hence, PO-1 inhibits an early step of metastasis, impairs unrestricted proliferation and induces apoptosis at low micromolar concentrations. These results warrant further testing in vivo to challenge the potential of PO-1 as novel lead compound.

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

    NASA Astrophysics Data System (ADS)

    Sandrock, Alfred W.; Matthew, William D.

    1987-10-01

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

  19. Assessment of chemical effects on neurite outgrowth in PC12 cells using high content screening

    EPA Science Inventory

    Identification of chemicals that pose a hazard to the developing nervous system is the first step in reducing human exposure and preventing health risks to infants and children. In response to the need for more efficient methods to identify potential developmental neurotoxicants,...

  20. Developmental neurotoxicity testing in vitro: Models for assessing chemical effects on neurite outgrowth

    EPA Science Inventory

    In vitro models may be useful for the rapid toxicological screening of large numbers of chemicals for their potential to produce toxicity. Such screening could facilitate prioritization of resources needed for in vivo toxicity testing towards those chemicals most likely to resul...

  1. Surface-Modified Nanofibrous Biomaterial Bridge for the Enhancement and Control of Neurite Outgrowth

    DTIC Science & Technology

    2010-12-22

    K . B . Mycek, Q. Chen, S . Samuel, E. L. Feldman, and D. C. Martin, J . Biomed. Mater. Res. Part A...Gandhi, F. K . Ko, and P. I . Lelkes, J . Biomed. Mater. Res. Part A 79A, 963 2006. 30E. Schnell, K . Klinkhammer, S . Balzer, G. Brook, D. Klee, P. Dalton...Biomaterials 29, 3574 2008. 34C. E. Ayres, B . S . Jha, H . Meredith, J . R. Bowman, G. L. Bowlin, S . C. Henderson, and D. G. Simpson, J .

  2. Endpoints for Neural Connectivity Including Neurite Outgrowth, Synapse Formation, and Function

    EPA Science Inventory

    A strategy for alternative methods for developmental neurotoxicity testing (DNT) focuses on assessment of chemical effects on conserved neurodevelopmental processes. The development of the brain is an integrated series of steps from the commitment of embryonic cells to become neu...

  3. Neurite outgrowth inhibitor Nogo-A establishes spatial segregation and extent of oligodendrocyte myelination

    PubMed Central

    Chong, S. Y. Christin; Rosenberg, Sheila S.; Fancy, Stephen P. J.; Zhao, Chao; Shen, Yun-An A.; Hahn, Angela T.; McGee, Aaron W.; Xu, Xiaomei; Zheng, Binhai; Zhang, Li I.; Rowitch, David H.; Franklin, Robin J. M.; Lu, Q. Richard; Chan, Jonah R.

    2012-01-01

    A requisite component of nervous system development is the achievement of cellular recognition and spatial segregation through competition-based refinement mechanisms. Competition for available axon space by myelinating oligodendrocytes ensures that all relevant CNS axons are myelinated properly. To ascertain the nature of this competition, we generated a transgenic mouse with sparsely labeled oligodendrocytes and establish that individual oligodendrocytes occupying similar axon tracts can greatly vary the number and lengths of their myelin internodes. Here we show that intercellular interactions between competing oligodendroglia influence the number and length of myelin internodes, referred to as myelinogenic potential, and identify the amino-terminal region of Nogo-A, expressed by oligodendroglia, as necessary and sufficient to inhibit this process. Exuberant and expansive myelination/remyelination is detected in the absence of Nogo during development and after demyelination, suggesting that spatial segregation and myelin extent is limited by microenvironmental inhibition. We demonstrate a unique physiological role for Nogo-A in the precise myelination of the developing CNS. Maximizing the myelinogenic potential of oligodendrocytes may offer an effective strategy for repair in future therapies for demyelination. PMID:22160722

  4. Neurite outgrowth inhibitor Nogo-A establishes spatial segregation and extent of oligodendrocyte myelination.

    PubMed

    Chong, S Y Christin; Rosenberg, Sheila S; Fancy, Stephen P J; Zhao, Chao; Shen, Yun-An A; Hahn, Angela T; McGee, Aaron W; Xu, Xiaomei; Zheng, Binhai; Zhang, Li I; Rowitch, David H; Franklin, Robin J M; Lu, Q Richard; Chan, Jonah R

    2012-01-24

    A requisite component of nervous system development is the achievement of cellular recognition and spatial segregation through competition-based refinement mechanisms. Competition for available axon space by myelinating oligodendrocytes ensures that all relevant CNS axons are myelinated properly. To ascertain the nature of this competition, we generated a transgenic mouse with sparsely labeled oligodendrocytes and establish that individual oligodendrocytes occupying similar axon tracts can greatly vary the number and lengths of their myelin internodes. Here we show that intercellular interactions between competing oligodendroglia influence the number and length of myelin internodes, referred to as myelinogenic potential, and identify the amino-terminal region of Nogo-A, expressed by oligodendroglia, as necessary and sufficient to inhibit this process. Exuberant and expansive myelination/remyelination is detected in the absence of Nogo during development and after demyelination, suggesting that spatial segregation and myelin extent is limited by microenvironmental inhibition. We demonstrate a unique physiological role for Nogo-A in the precise myelination of the developing CNS. Maximizing the myelinogenic potential of oligodendrocytes may offer an effective strategy for repair in future therapies for demyelination.

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

    PubMed Central

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

    2015-01-01

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

  6. Nuclear kinesis, neurite sprouting and abnormal axonal projections of cone photoreceptors in the aged and AMD-afflicted human retina.

    PubMed

    Pow, David V; Sullivan, Robert K P

    2007-05-01

    Tissues often respond to damage by recapitulating developmental programs. We have investigated whether anatomical signs of developmental recapitulation are evident in cone photoreceptors of the aged and AMD-afflicted human retina. Radial migration of cell nuclei mediated by microtubules is a characteristic feature of cells in the developing retina. Similarly, neurite outgrowth is a feature of developing neurons. We have examined whether nuclear kinesis and neurite outgrowth from cone photoreceptors is evident. Calbindin-positive cone photoreceptor nuclei are normally positioned as a single layer of somata at the outer border of the outer nuclear layer. In AMD-afflicted retinae, many nuclei are translocated, with some somata abutting the outer plexiform layer (OPL) and others outside the outer limiting membrane whilst many nuclei are present at intermediate levels. The axonal processes of many cones were also aberrant, displaying tortuous pathways as they projected to the OPL, with occasional evidence for bifurcation at points where the axon changed direction. We suggest that tangential extension of collateral neurites and the rapid retraction of the original process may give rise to the tortuous axonal projections observed. Since microtubules are key mediators of both neurite extension and nuclear kinesis we examined expression of microtubule associated protein 2 (MAP2) which is an important regulator of neurite extension. The strong expression of MAP2 observed in those cells with aberrant morphologies supports the notion that abnormal microtubule-mediated remodelling events are present in the AMD retina and to a lesser extent in normal aged retinas, allowing cone photoreceptors to recapitulate two key features of development.

  7. VANG-1 and PRKL-1 Cooperate to Negatively Regulate Neurite Formation in Caenorhabditis elegans

    PubMed Central

    Su, Anna; Imai, Janice H.; Colavita, Antonio

    2011-01-01

    Neuritogenesis is a critical early step in the development and maturation of neurons and neuronal circuits. While extracellular directional cues are known to specify the site and orientation of nascent neurite formation in vivo, little is known about the genetic pathways that block inappropriate neurite emergence in order to maintain proper neuronal polarity. Here we report that the Caenorhabditis elegans orthologues of Van Gogh (vang-1), Prickle (prkl-1), and Dishevelled (dsh-1), core components of planar cell polarity (PCP) signaling, are required in a subset of peripheral motor neurons to restrict neurite emergence to a specific organ axis. In loss-of-function mutants, neurons display supernumerary neurites that extend inappropriately along the orthogonal anteroposterior (A/P) body axis. We show that autonomous and non-autonomous gene activities are required early and persistently to inhibit the formation or consolidation of growth cone protrusions directed away from organ precursor cells. Furthermore, prkl-1 overexpression is sufficient to suppress neurite formation and reorient neuronal polarity in a vang-1– and dsh-1–dependent manner. Our findings suggest a novel role for a PCP–like pathway in maintaining polarized neuronal morphology by inhibiting neuronal responses to extrinsic or intrinsic cues that would otherwise promote extraneous neurite formation. PMID:21912529

  8. Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth

    PubMed Central

    Awal, Mehraj R.; Shay, James; McLoed, Melissa M.; Mazur, Eric; Gabel, Christopher V.

    2016-01-01

    During development, a neuron transitions from a state of rapid growth to a stable morphology, and neurons within the adult mammalian CNS lose their ability to effectively regenerate in response to injury. Here, we identify a novel form of neuronal regeneration, which is remarkably independent of DLK-1/DLK, KGB-1/JNK, and other MAPK signaling factors known to mediate regeneration in Caenorhabditis elegans, Drosophila, and mammals. This DLK-independent regeneration in C. elegans has direct genetic and molecular links to a well-studied form of endogenous activity-dependent ectopic axon outgrowth in the same neuron type. Both neuron outgrowth types are triggered by physical lesion of the sensory dendrite or mutations disrupting sensory activity, calcium signaling, or genes that restrict outgrowth during neuronal maturation, such as SAX-1/NDR kinase or UNC-43/CaMKII. These connections suggest that ectopic outgrowth represents a powerful platform for gene discovery in neuronal regeneration. Moreover, we note numerous similarities between C. elegans DLK-independent regeneration and lesion conditioning, a phenomenon producing robust regeneration in the mammalian CNS. Both regeneration types are triggered by lesion of a sensory neurite via reduction of neuronal activity and enhanced by disrupting L-type calcium channels or elevating cAMP. Taken as a whole, our study unites disparate forms of neuronal outgrowth to uncover fresh molecular insights into activity-dependent control of the adult nervous system’s intrinsic regenerative capacity. PMID:27078101

  9. Insulin signaling regulates neurite growth during metamorphic neuronal remodeling

    PubMed Central

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

    2014-01-01

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

  10. Scale of Carbon Nanomaterials Affects Neural Outgrowth and Adhesion

    PubMed Central

    Franca, Eric; Jao, PitFee; Fang, Sheng-Po; Alagapan, Sankaraleengam; Pan, Liangbin; Yoon, Jung Hae; Yoon, Yong-Kyu ‘YK’

    2016-01-01

    Carbon nanomaterials have become increasingly popular microelectrode materials for neuroscience applications. Here we study how the scale of carbon nanotubes and carbon nanofibers affect neural viability, outgrowth, and adhesion. Carbon nanotubes were deposited on glass coverslips via a layer-by-layer method with polyethylenimine (PEI). Carbonized nanofibers were fabricated by electrospinning SU-8 and pyrolyzing the nanofiber depositions. Additional substrates tested were carbonized and SU-8 thin films and SU-8 nanofibers. Surfaces were O2-plasma treated, coated with varying concentrations of PEI, seeded with E18 rat cortical cells, and examined at 3, 4, and 7 days in vitro (DIV). Neural adhesion was examined at 4 DIV utilizing a parallel plate flow chamber. At 3 DIV, neural viability was lower on the nanofiber and thin film depositions treated with higher PEI concentrations which corresponded with significantly higher zeta potentials (surface charge); this significance was drastically higher on the nanofibers suggesting that the nanostructure may collect more PEI molecules, causing increased toxicity. At 7 DIV, significantly higher neurite outgrowth was observed on SU-8 nanofiber substrates with nanofibers a significant fraction of a neuron’s size. No differences were detected for carbonized nanofibers or carbon nanotubes. Both carbonized and SU-8 nanofibers had significantly higher cellular adhesion post-flow in comparison to controls whereas the carbon nanotubes were statistically similar to control substrates. These data suggest a neural cell preference for larger-scale nanomaterials with specific surface treatments. These characteristics could be taken advantage of in the future design and fabrication of neural microelectrodes. PMID:26829799

  11. The Caenorhabditis elegans Ephrin EFN-4 Functions Non-cell Autonomously with Heparan Sulfate Proteoglycans to Promote Axon Outgrowth and Branching

    PubMed Central

    Schwieterman, Alicia A.; Steves, Alyse N.; Yee, Vivian; Donelson, Cory J.; Bentley, Melissa R.; Santorella, Elise M.; Mehlenbacher, Taylor V.; Pital, Aaron; Howard, Austin M.; Wilson, Melissa R.; Ereddia, Danielle E.; Effrein, Kelsie S.; McMurry, Jonathan L.; Ackley, Brian D.; Chisholm, Andrew D.; Hudson, Martin L.

    2016-01-01

    The Eph receptors and their cognate ephrin ligands play key roles in many aspects of nervous system development. These interactions typically occur within an individual tissue type, serving either to guide axons to their terminal targets or to define boundaries between the rhombomeres of the hindbrain. We have identified a novel role for the Caenorhabditis elegans ephrin EFN-4 in promoting primary neurite outgrowth in AIY interneurons and D-class motor neurons. Rescue experiments reveal that EFN-4 functions non-cell autonomously in the epidermis to promote primary neurite outgrowth. We also find that EFN-4 plays a role in promoting ectopic axon branching in a C. elegans model of X-linked Kallmann syndrome. In this context, EFN-4 functions non-cell autonomously in the body-wall muscle and in parallel with HS modification genes and HSPG core proteins. This is the first report of an epidermal ephrin providing a developmental cue to the nervous system. PMID:26645816

  12. The Effect of Surface Modification of Aligned Poly-L-Lactic Acid Electrospun Fibers on Fiber Degradation and Neurite Extension

    PubMed Central

    Schaub, Nicholas J.; Le Beux, Clémentine; Miao, Jianjun; Linhardt, Robert J.; Alauzun, Johan G.; Laurencin, Danielle; Gilbert, Ryan J.

    2015-01-01

    The surface of aligned, electrospun poly-L-lactic acid (PLLA) fibers was chemically modified to determine if surface chemistry and hydrophilicity could improve neurite extension from chick dorsal root ganglia. Specifically, diethylenetriamine (DTA, for amine functionalization), 2-(2-aminoethoxy)ethanol (AEO, for alcohol functionalization), or GRGDS (cell adhesion peptide) were covalently attached to the surface of electrospun fibers. Water contact angle measurements revealed that surface modification of electrospun fibers significantly improved fiber hydrophilicity compared to unmodified fibers (p < 0.05). Scanning electron microscopy (SEM) of fibers revealed that surface modification changed fiber topography modestly, with DTA modified fibers displaying the roughest surface structure. Degradation of chemically modified fibers revealed no change in fiber diameter in any group over a period of seven days. Unexpectedly, neurites from chick DRG were longest on fibers without surface modification (1651 ± 488 μm) and fibers containing GRGDS (1560 ± 107 μm). Fibers modified with oxygen plasma (1240 ± 143 μm) or DTA (1118 ± 82 μm) produced shorter neurites than the GRGDS or unmodified fibers, but were not statistically shorter than unmodified and GRGDS modified fibers. Fibers modified with AEO (844 ± 151 μm) were significantly shorter than unmodified and GRGDS modified fibers (p<0.05). Based on these results, we conclude that fiber hydrophilic enhancement alone on electrospun PLLA fibers does not enhance neurite outgrowth. Further work must be conducted to better understand why neurite extension was not improved on more hydrophilic fibers, but the results presented here do not recommend hydrophilic surface modification for the purpose of improving neurite extension unless a bioactive ligand is used. PMID:26340351

  13. An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

    PubMed Central

    Liebau, Stefan; Steinestel, Julie; Linta, Leonhard; Kleger, Alexander; Storch, Alexander; Schoen, Michael; Steinestel, Konrad; Proepper, Christian; Bockmann, Juergen; Schmeisser, Michael J.; Boeckers, Tobias M.

    2011-01-01

    Background The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell. Principal Findings We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners. Conclusions Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins. PMID:21464958

  14. Detection of neuritic plaques in Alzheimer’s disease by magnetic resonance microscopy

    PubMed Central

    Benveniste, Helene; Einstein, Gillian; Kim, Katie R.; Hulette, Christine; Johnson, G. Allan

    1999-01-01

    Magnetic resonance microscopy (MRM) theoretically provides the spatial resolution and signal-to-noise ratio needed to resolve neuritic plaques, the neuropathological hallmark of Alzheimer’s disease (AD). Two previously unexplored MR contrast parameters, T2* and diffusion, are tested for plaque-specific contrast to noise. Autopsy specimens from nondemented controls (n = 3) and patients with AD (n = 5) were used. Three-dimensional T2* and diffusion MR images with voxel sizes ranging from 3 × 10−3 mm3 to 5.9 × 10−5 mm3 were acquired. After imaging, specimens were cut and stained with a microwave king silver stain to demonstrate neuritic plaques. From controls, the alveus, fimbria, pyramidal cell layer, hippocampal sulcus, and granule cell layer were detected by either T2* or diffusion contrast. These structures were used as landmarks when correlating MRMs with histological sections. At a voxel resolution of 5.9 × 10−5 mm3, neuritic plaques could be detected by T2*. The neuritic plaques emerged as black, spherical elements on T2* MRMs and could be distinguished from vessels only in cross-section when presented in three dimension. Here we provide MR images of neuritic plaques in vitro. The MRM results reported provide a new direction for applying this technology in vivo. Clearly, the ability to detect and follow the early progression of amyloid-positive brain lesions will greatly aid and simplify the many possibilities to intervene pharmacologically in AD. PMID:10570201

  15. Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells

    PubMed Central

    Hirose, Masaya; Ishizaki, Toshimasa; Watanabe, Naoki; Uehata, Masayoshi; Kranenburg, Onno; Moolenaar, Wouter H.; Matsumura, Fumio; Maekawa, Midori; Bito, Haruhiko; Narumiya, Shuh

    1998-01-01

    A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells. PMID:9647654

  16. Inhibitory effects of Eleutherococcus senticosus extracts on amyloid beta(25-35)-induced neuritic atrophy and synaptic loss.

    PubMed

    Tohda, Chihiro; Ichimura, Mahoko; Bai, Yanjing; Tanaka, Ken; Zhu, Shu; Komatsu, Katsuko

    2008-07-01

    Neurons with atrophic neurites may remain alive and therefore may have the potential to regenerate even when neuronal death has occurred in some parts of the brain. This study aimed to explore effects of drugs that can facilitate the regeneration of neurites and the reconstruction of synapses even in severely damaged neurons. We investigated the effects of Eleutherococcus senticosus extracts on the regeneration of neurites and the reconstruction of synapses in rat cultured cortical neurons damaged by amyloid beta (Abeta)(25-35). Treatment with Abeta(25-35) (10 microM) induced axonal and dendritic atrophies and synaptic loss in cortical neurons. Subsequent treatment with the methanol extract and the water extract of E. senticosus (10 - 1000 ng/ml) resulted in significant axonal and dendritic regenerations and reconstruction of neuronal synapses. Co-application of the extract and Abeta(25-35) attenuated Abeta(25-35)-induced neuronal death. We investigated neurite outgrowth activities of eleutherosides B and E and isoflaxidin, which are known as major compounds in E. senticosus. Although eleutheroside B protected against Abeta(25-35)-induced dendritic and axonal atrophies, the activities of eleutheroside E and isofraxidin were less than that of eleutheroside B. Although the contents of these three compounds in the water extract were less than in the methanol extract, restoring activities against neuronal damages were not different between the two extracts. In conclusion, extracts of E. senticosus protect against neuritic atrophy and cell death under Abeta treatment, and one of active constituents may be eleutheroside B.

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

    Kador, Karl E.; Grogan, Shawn P.; Dorthé, Erik W.; Venugopalan, Praseeda; Malek, Monisha F.

    2016-01-01

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

  19. NIF (neurite-inducing factor): a novel peptide inducing neurite formation in PC12 cells.

    PubMed

    Wagner, J A

    1986-01-01

    Neurite-inducing factor (NIF) is a novel protein that has been partially purified from mouse submaxillary glands. NIF induces neurite formation in PC12 pheochromocytoma cells, and the NIF-induced neurites are indistinguishable from NGF-induced neurites in both their morphology and the time course of their formation. Neurite-inducing activity can be recovered at a position corresponding to a molecular weight of 20,000 Da after fractionation of partially purified preparations via SDS-PAGE. Partially purified preparations of NIF are about half as potent as pure beta NGF, and since the neurite-inducing activity does not correspond to any of the major proteins in this fraction, specific activity of purified NIF will probably be significantly greater than the 60 ng/ml found for our partially purified material. NIF is distinct from beta NGF by four criteria: (1) antibodies to beta NGF can block the activity of beta NGF, but not the activity of NIF; (2) beta NGF can induce ornithine decarboxylase (ODC) in PC12 cells at concentrations significantly below those required to induce neurites, while NIF induces ODC only at concentrations greatly in excess of those required to induce neurite formation; (3) by the criterion of SDS-PAGE, there is insufficient beta NGF in our partially purified preparations of NIF to explain the biological activity of this fraction; and (4) the biological activity of NIF has a molecular weight (20,000 Da) that is distinct from beta NGF (13,000 Da). We conclude that NIF is probably a novel peptide that is very active in promoting morphological differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Nerve growth factor-immobilized polypyrrole: Bioactive electrically conducting polymer for enhanced neurite extension

    PubMed Central

    Gomez, Natalia; Schmidt, Christine E.

    2010-01-01

    Biomaterials that present multiple stimuli are attractive for a number of biomedical applications. In particular, electrical and biological cues are important factors to include in interfaces with neurons for applications such as nerve conduits and neural probes. Here, we report the combination of these two stimuli, by immobilizing nerve growth factor (NGF) on the surface of the electrically conducting polymer polypyrrole (PPy). NGF was immobilized using an intermediate linker provided by a layer of polyallylamine conjugated to an arylazido functional group. Upon exposure to UV light and activation of the azido groups, NGF was fixed to the substrate. Three different surface concentrations were obtained (0.21–0.98 ng/mm2) and similar levels of neurite extension were observed on immobilized NGF as with soluble NGF. Additionally, electrical stimulation experiments were conducted with the modified polymer and revealed a 50% increase in neurite outgrowth in PC12 cells compared to experiments without electrical stimulation. This novel modification of PPy provides both electrical and biological stimulation, by presenting tethered growth factors and only producing a small decrease in the material's properties (conductivity ~10 S cm−1) when compared to other modification techniques (conductivity ~10−3–10−6 S cm−1. PMID:17111407

  1. ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

    SciTech Connect

    Han, Jeong-Ho; Yu, Tae-Hoon; Ryu, Hyun-Hee; Jun, Mi-Hee; Ban, Byung-Kwan; Jang, Deok-Jin; Lee, Jin-A

    2013-08-01

    Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.

  2. Formation of polarity convergences underlying shoot outgrowths

    PubMed Central

    Abley, Katie; Sauret-Güeto, Susanna; Marée, Athanasius FM; Coen, Enrico

    2016-01-01

    The development of outgrowths from plant shoots depends on formation of epidermal sites of cell polarity convergence with high intracellular auxin at their centre. A parsimonious model for generation of convergence sites is that cell polarity for the auxin transporter PIN1 orients up auxin gradients, as this spontaneously generates convergent alignments. Here we test predictions of this and other models for the patterns of auxin biosynthesis and import. Live imaging of outgrowths from kanadi1 kanadi2 Arabidopsis mutant leaves shows that they arise by formation of PIN1 convergence sites within a proximodistal polarity field. PIN1 polarities are oriented away from regions of high auxin biosynthesis enzyme expression, and towards regions of high auxin importer expression. Both expression patterns are required for normal outgrowth emergence, and may form part of a common module underlying shoot outgrowths. These findings are more consistent with models that spontaneously generate tandem rather than convergent alignments. DOI: http://dx.doi.org/10.7554/eLife.18165.001 PMID:27478985

  3. Exploiting Differential Surface Display of Chondroitin Sulfate Variants for Directing Neuronal Outgrowth

    PubMed Central

    Swarup, Vimal P.; Hsiao, Tony W.; Zhang, Jianxing; Prestwich, Glenn D.; Kuberan, Balagurunathan; Hlady, Vladimir

    2014-01-01

    Chondroitin sulfate (CS) proteoglycans (CSPGs) are known to be primary inhibitors of neuronal regeneration at scar sites. However, a variety of CSPGs are also involved in neuronal growth and guidance during other physiological stages. Sulfation patterns of CS chains influence their interactions with various growth factors in the central nervous system (CNS), thus influencing neuronal growth, inhibition, and pathfinding. This report demonstrates the use of differentially sulfated CS chains for neuronal navigation. Surface-immobilized patterns of CS glycosaminoglycan chains were used to determine neuronal preference toward specific sulfations of five CS variants: CS-A, CS-B (dermatan sulfate), CS-C, CS-D, and CS-E. Neurons preferred CS-A, CS-B, and CS-E and avoided CS-C containing lanes. In addition, significant alignment of neurites was observed using underlying lanes containing CS-A, CS-B, and CS-E chains. To utilize differential preference of neurons toward the CS variants, a binary combinations of CS chains were created by backfilling a neuro-preferred CS variant between the microcontact printed lanes of CS-C stripes, which are avoided by neurons. The neuronal outgrowth results demonstrate for the first time that a combination of sulfation variants of CS chains without any protein component of CSPG is sufficient for directing neuronal outgrowth. Biomaterials with surface immobilized GAG chains could find numerous applications as bridging devices for tackling CNS injuries where directional growth of neurons is critical for recovery. PMID:23947484

  4. C. elegans fmi-1/flamingo and Wnt pathway components interact genetically to control the anteroposterior neurite growth of the VD GABAergic neurons.

    PubMed

    Huarcaya Najarro, Elvis; Ackley, Brian D

    2013-05-01

    Directed axonal growth is essential to establish neuronal networks. During the early development of the VD neurons, an anterior neurite that will become the VD axon extends along the anteroposterior (A/P) axis in the ventral nerve cord (VNC) in Caenorhabditis elegans. Little is known about the cellular and molecular mechanisms that are important for correct neurite growth in the VNC. In fmi-1/flamingo mutant animals, we observed that some postembryonically born VD neurons had a posterior neurite instead of a normal anterior neurite, which caused aberrant VD commissure patterning along the A/P axis. In addition, VD anterior neurites had underextension defects in the VNC in fmi-1 animals, whereas VD commissure growth along the dorsoventral (D/V) axis occurred normally in these animals, suggesting that fmi-1 is important for neurite growth along the A/P axis but not the D/V axis. We also uncovered unknown details of the early development of the VD neurons, indicating that the neurite defects arose during their early development. Interestingly, though fmi-1 is present at this time in the VNC, we did not observe FMI-1 in the VD neurons themselves, suggesting that fmi-1 might be working in a cell non-autonomous fashion. Furthermore, fmi-1 appears to be working in a novel pathway, independently from the planar cell polarity pathway and in parallel to lin-17/frizzled and dsh-1/dishevelled, to determine the direction of neurite growth. Our findings indicate that redundant developmental pathways regulate neurite growth in the VNC in C. elegans.

  5. Lysophosphatidic acid induces osteocyte dendrite outgrowth.

    PubMed

    Karagiosis, Sue A; Karin, Norman J

    2007-05-25

    Osteocytes elaborate an extensive mechanosensory network in bone matrix and communicate intercellularly via gap junctions established at dendrite termini. We developed a method to measure osteocyte dendritogenesis in vitro using a modified transwell assay and determined that the lipid growth factor lysophosphatidic acid (LPA) is a potent stimulator of dendrite outgrowth in MLO-Y4 osteocytes. The stimulatory effects were dose-dependent with maximal outgrowth observed within a physiological range of LPA. LPA-treated osteocytes exhibited distinct rearrangements of the actin cytoskeleton and a more stellate morphology than control cells. LPA also promoted osteocyte chemotaxis, suggesting a shared molecular mechanism between dendrite outgrowth and cell motility. The LPA-induced increase in dendrite formation was blocked by the specific LPA-receptor antagonist Ki16425 and by pertussis toxin. Bone cells in vivo encounter platelet-derived LPA in regions of bone damage, and we postulate that this lipid factor is important for re-establishing osteocyte connectivity during fracture repair.

  6. Effects of Reducing Suppressors of Cytokine Signaling-3 (SOCS3) Expression on Dendritic Outgrowth and Demyelination after Spinal Cord Injury

    PubMed Central

    Park, Keun Woo; Lin, Ching-Yi; Li, Kevin; Lee, Yu-Shang

    2015-01-01

    Suppressors of cytokine signaling-3 (SOCS3) is associated with limitations of nerve growth capacity after injury to the central nervous system. Although genetic manipulations of SOCS3 can enhance axonal regeneration after optic injury, the role of SOCS3 in dendritic outgrowth after spinal cord injury (SCI) is still unclear. The present study investigated the endogenous expression of SOCS3 and its role in regulating neurite outgrowth in vitro. Interleukin-6 (IL-6) induces SOCS3 expression at the mRNA and protein levels in neuroscreen-1 (NS-1) cells. In parallel to SOCS3 expression, IL-6 induced tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) in NS-1 cells. Lentiviral delivery of short hairpin RNA (shSOCS3) (Lenti-shSOCS3) to decrease SOCS3 expression into NS-1 cells enhanced IL-6-induced tyrosine phosphorylation of STAT3 (P-STAT3 Tyr705) and promoted neurite outgrowth. In addition, we determined if reduction of SOCS3 expression by microinjection of Lenti-shSOCS3 into spinal cord enhances dendrite outgrowth in spinal cord neurons after SCI. Knocking down of SOCS3 in spinal cord neurons with Lenti-shSOCS3 increased complete SCI-induced P-STAT3 Tyr705. Immunohistochemical analysis showed that complete SCI induced a significant reduction of microtubule association protein 2-positive (MAP-2+) dendrites in the gray and white matter at 1 and 4 weeks after injury. The SCI-induced reduction of MAP-2+ dendrites was inhibited by infection with Lenti-shSOCS3 in areas both rostral and caudal to the lesion at 1 and 4 weeks after complete SCI. Furthermore, shSOCS3 treatment enhanced up-regulation of growth associated protein-43 (GAP-43) expression, which co-localized with MAP-2+ dendrites in white matter and with MAP-2+ cell bodies in gray matter, indicating Lenti-shSOCS3 may induce dendritic regeneration after SCI. Moreover, we demonstrated that Lenti-shSOCS3 decreased SCI-induced demyelination in white matter of spinal cord both rostral and

  7. INFLUENCE OF DIFFERENT INCUBATOR MODELS ON MAGNETIC FIELD-INDUCED CHANGES IN NEURITE OUTGROWTH IN PC-12 CELLS

    EPA Science Inventory

    OBJECTIVE: Devise a method to standardize responses of cells to MF-exposure in different incubator environments. METHODS: We compared the cell responses to generated MF in a standard cell-culture incubator (Forma, model #3158) with cell responses to the same exposure when a mu-m...

  8. Screening for Chemical Effects on Neuronal Proliferation and Neurite Outgrowth Using High-Content/High-Throughput Microscopy

    EPA Science Inventory

    The need to develop novel screening methods for developmental neurotoxicity in order to alleviate the demands of cost, time, and animals required for in vivo toxicity studies is well recognized. Accordingly, the U.S. EPA launched the ToxCast research program in 2007 to develop c...

  9. MANGANESE INDUCES NEURITE OUTGROWTH IN PC12 CELLS VIA UPREGULATION OF ALPHA(V) INTEGRINS. (R826248)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  10. ASSESSMENT OF CHEMICAL EFFECTS ON NEURITE OUTGROWTH, NEURONAL POLARIZATION AND SYNAPTOGENESIS IN RAT CORTICAL NEURONS USING HIGH CONTENT IMAGE ANALYSIS

    EPA Science Inventory

    There is a need for efficient, cost-effective methods for screening and prioritization of potential developmental neurotoxicants. One approach uses in vitro cell culture models that can recapitulate the critical processes of nervous system development. In vitro, primary cultures ...

  11. COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM

    EPA Science Inventory

    A major challenge facing the Environmental Protection Agency is the development of high-throughput screening assays amendable to resource-efficient developmental neurotoxicity for chemical screening and toxicity prioritization. One approach uses in vitro, cell-based assays which...

  12. The Effects of Target Skeletal Muscle Cells on Dorsal Root Ganglion Neuronal Outgrowth and Migration In Vitro

    PubMed Central

    Zhang, Weiwei; Li, Zhenzhong

    2013-01-01

    Targets of neuronal innervations play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. During development, neurons extend axons to their targets, and then their survival become dependent on the trophic substances secreted by their target cells. Sensory endings were present on myoblasts, myotubes, and myofibers in all intrafusal bundles regardless of age. The interdependence of sensory neurons and skeletal muscle (SKM) cells during both embryonic development and the maintenance of the mature functional state has not been fully understood. In the present study, neuromuscular cocultures of organotypic dorsal root ganglion (DRG) explants and dissociate SKM cells were established. Using this culture system, the morphological relationship between DRG neurons and SKM cells, neurites growth and neuronal migration were investigated. The migrating neurons were determined by fluorescent labeling of microtubule-associated protein-2 (MAP-2) and neurofilament 200 (NF-200) or growth-associated protein 43 (GAP-43). The expression of NF-200 and GAP-43 and their mRNAs was evaluated by Western blot assay and real time-PCR analysis. The results reveal that DRG explants showed more dense neurites outgrowth in neuromuscular cocultures as compared with that in the culture of DRG explants alone. The number of total migrating neurons (the MAP-2-expressing neurons) and the percentage NF-200-immunoreactive (IR) and GAP-43-IR neurons increased significantly in the presence of SKM cells. The levels of NF-200 and GAP-43 and their mRNAs increased significantly in neuromuscular cocultures as compared with that in the culture of DRG explants alone. These results suggested that target SKM cells play an important role in regulating neuronal protein synthesis, promoting neuritis outgrowth and neuronal migration of DRG explants in vitro. These results not only provide new clues for a better understanding of the association of SKM cells with

  13. C. elegans fmi-1/flamingo and Wnt pathway components interact genetically to control the anteroposterior neurite growth of the VD GABAergic neurons.

    PubMed

    Ackley, Brian D

    2013-07-01

    During development, multiple environmental cues, e.g., growth factors, cell adhesion molecules, etc., interact to influence the pattern of outgrowth of axons and dendrites in a cell-specific fashion. As a result, individual neurons may receive similar signals, but make unique choices, leading to distinct wiring within the nervous system. C. elegans has been useful in identifying molecular cues that influence neuronal development, as well as the downstream mechanisms that allow individual neurons to make cell-specific responses. Recently, we described a role for the conserved cadherin domain-containing protein, FMI-1/flamingo, in multiple stages of neural development in C. elegans. During the initial phase of neurite outgrowth, FMI-1 seems to have a relatively cell-specific effect on the VD neurons to promote the initial neurite formed to grow toward the anterior. In this capacity, FMI-1 appears to work coordinately with at least two Wnt ligands, EGL-20 and LIN-44, and multiple downstream Wnt signaling components (including LIN-17/Frizzled, DSH-1/Disheveled, and BAR-1/β-catenin). Here I will discuss some of the ideas we considered about how FMI-1 could affect neurons as they acquire their morphology during development.

  14. Coordinate expression of IGF-I and its receptor during limb outgrowth.

    PubMed

    Geduspan, J S; Padanilam, B J; Solursh, M

    1992-09-01

    The morphogenetic mechanisms involved in shaping the embyro are largely unknown. Previous studies from this laboratory suggest that the mesonephros promotes limb outgrowth in ovo in the chicken embryo and might be involved in early limb morphogenesis, since damage to the mesonephros results in truncated limbs. In limb bud organ cultures, the presence of the mesonephros promotes cartilage formation. This effect can be reproduced by exogenous IGF-I or prevented by blocking antibody to IGF-I. In order to examine the hypothesis that mesonephros-derived IGF-I is involved in the early morphogenesis of the limb, we examined the spatial and temporal expression of IGF-I and type I receptor for IGF by in situ hybridization at stages when the onset of limb development occurs. The results show that neither transcript is detected at stage 13, prior to the appearance of the limb bud; but both transcripts are detected in the mesonephros at stage 14, an early stage in limb outgrowth. The hybridization signal in the mesonephros for both transcripts increases with development and signal was codistributed as well. At stage 18 the level of receptor transcripts detected in the flank relative to the limb decreased. Thus, the temporal and spatial patterns of expression of IGF-I and its receptor are consistent with their involvement in the initiation of limb outgrowth and support the model that localized expression of a growth factor and its receptor can be involved in shaping the embryo.

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

    PubMed

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

    2015-05-10

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

  16. Centella asiatica accelerates nerve regeneration upon oral administration and contains multiple active fractions increasing neurite elongation in-vitro.

    PubMed

    Soumyanath, Amala; Zhong, Yong-Ping; Gold, Sandra A; Yu, Xiaolin; Koop, Dennis R; Bourdette, Dennis; Gold, Bruce G

    2005-09-01

    Axonal regeneration is important for functional recovery following nerve damage. Centella asiatica Urban herb, also known as Hydrocotyle asiatica L., has been used in Ayurvedic medicine for centuries as a nerve tonic. Here, we show that Centella asiatica ethanolic extract (100 microg mL-1) elicits a marked increase in neurite outgrowth in human SH-SY5Y cells in the presence of nerve growth factor (NGF). However, a water extract of Centella was ineffective at 100 microg mL-1. Sub-fractions of Centella ethanolic extract, obtained through silica-gel chromatography, were tested (100 microg mL-1) for neurite elongation in the presence of NGF. Greatest activity was found with a non-polar fraction (GKF4). Relatively polar fractions (GKF10 to GKF13) also showed activity, albeit less than GKF4. Thus, Centella contains more than one active component. Asiatic acid (AA), a triterpenoid compound found in Centella ethanolic extract and GKF4, showed marked activity at 1 microM (microg mL-1). AA was not present in GKF10 to GKF13, further indicating that other active components must be present. Neurite elongation by AA was completely blocked by the extracellular-signal-regulated kinase (ERK) pathway inhibitor PD 098059 (10 microM). Male Sprague-Dawley rats given Centella ethanolic extract in their drinking water (300-330 mg kg-1 daily) demonstrated more rapid functional recovery and increased axonal regeneration (larger calibre axons and greater numbers of myelinated axons) compared with controls, indicating that the axons grew at a faster rate. Taken together, our findings indicate that components in Centella ethanolic extract may be useful for accelerating repair of damaged neurons.

  17. Brain-derived neurotrophic factor promotes neurite growth and survival of antennal lobe neurons in brain from the silk moth, Bombyx mori in vitro.

    PubMed

    Kim, Jin Hee; Sung, Dong Kyung; Park, Chan Woo; Park, Hun Hee; Park, Cheolin; Jeon, Soung-Hoo; Kang, Pil Don; Kwon, O-Yu; Lee, Bong Hee

    2005-03-01

    This study was conducted to investigate effects of brain-derived neurotrophic factor on the neurite growth and the survival rate of antennal lobe neurons in vitro, and secretion of brain-derived neurotrophic factor-like neuropeptide from brain into hemolymph in the silk moth, Bombyx mori. In primary culture of antennal lobe neurons with brain-derived neurotrophic factor, it promoted both a neurite extension of putative antennal lobe projection neurons and an outgrowth of branches from principal neurites of putative antennal interneurons with significance (p<0.05). Brain-derived neurotrophic factor also increased significantly a survival rate of antennal lobe neurons (p<0.05). Results from immunolabeling of brain and retrocerebral complex, and ELISA assay of hemolymph showed that brain-derived neurotrophic factor-like neuropeptide was synthesized by both median and lateral neurosecretory cells of brain, then transported to corpora allata for storage, and finally secreted into hemolymph for action. These results will provide valuable information for differentiation of invertebrate brain neurons with brain-derived neurotrophic factor.

  18. Expression of Rac1 alternative 3' UTRs is a cell specific mechanism with a function in dendrite outgrowth in cortical neurons.

    PubMed

    Braz, Sandra Oliveira; Cruz, Andrea; Lobo, Andrea; Bravo, Joana; Moreira-Ribeiro, Joana; Pereira-Castro, Isabel; Freitas, Jaime; Relvas, Joao B; Summavielle, Teresa; Moreira, Alexandra

    2017-03-06

    The differential expression of mRNAs containing tandem alternative 3' UTRs, achieved by mechanisms of alternative polyadenylation and post-transcriptional regulation, has been correlated with a variety of cellular states. In differentiated cells and brain tissues there is a general use of distal polyadenylation signals, originating mRNAs with longer 3' UTRs, in contrast with proliferating cells and other tissues such as testis, where most mRNAs contain shorter 3' UTRs. Although cell type and state are relevant in many biological processes, how these mechanisms occur in specific brain cell types is still poorly understood. Rac1 is a member of the Rho family of small GTPases with essential roles in multiple cellular processes, including cell differentiation and axonal growth. Here we used different brain cell types and tissues, including oligodendrocytes, microglia, astrocytes, cortical and hippocampal neurons, and optical nerve, to show that classical express mRNAs with alternative 3' UTRs differently, by gene- and cell- specific mechanisms. In particular, we show that Rac1 originate mRNA isoforms with longer 3' UTRs specifically during neurite growth of cortical, but not hippocampal neurons. Furthermore, we demonstrate that the longest Rac1 3' UTR is necessary for driving the mRNA to the neurites, and also for neurite outgrowth in cortical neurons. Our results indicate that the expression of Rac1 longer 3' UTR is a gene and cell-type specific mechanism in the brain, with a new physiological function in cortical neuron differentiation.

  19. Hepassocin is required for hepatic outgrowth during zebrafish hepatogenesis

    SciTech Connect

    Gao, Ming; Yan, Hui; Yin, Rong-Hua; Wang, Qiang; Zhan, Yi-Qun; Yu, Miao; Ge, Chang-Hui; Li, Chang-Yan; Wang, Xiao-Hui; Ge, Zhi-Qiang; Yang, Xiao-Ming

    2015-07-31

    Background & aims: Hepassocin (HPS) is a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage. In this paper, zebrafish were used to investigate the role of HPS in liver development. Methods and results: During zebrafish development, HPS expression is enriched in liver throughout hepatogenesis. Knockdown of HPS using its specific morpholino leads to a smaller liver phenotype. Further results showed that the HPS knockdown has no effect on the expression of the early endoderm marker gata6 and early hepatic marker hhex. In addition, results showed that the smaller-liver phenotype in HPS morphants was caused by suppression of cell proliferation, not induction of cell apoptosis. Conclusions: Current findings indicated that HPS is essential to the later stages of development in vertebrate liver organogenesis. - Highlights: • HPS is enriched in zebrafish liver and has strong similarities with other species. • Knocking down HPS with MOs results in small liver phenotype. • HPS depletion regulates liver outgrowth but not liver specification and budding. • HPS depletion causes hepatocyte proliferation arrest but not apoptosis induction.

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

    PubMed Central

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

    2016-01-01

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

  1. A novel fibroblast growth factor receptor family member promotes neuronal outgrowth and synaptic plasticity in aplysia.

    PubMed

    Pollak, Daniela D; Minh, Bui Quang; Cicvaric, Ana; Monje, Francisco J

    2014-11-01

    Fibroblast Growth Factor (FGF) Receptors (FGFRs) regulate essential biological processes, including embryogenesis, angiogenesis, cellular growth and memory-related long-term synaptic plasticity. Whereas canonical FGFRs depend exclusively on extracellular Immunoglobulin (Ig)-like domains for ligand binding, other receptor types, including members of the tropomyosin-receptor-kinase (Trk) family, use either Ig-like or Leucine-Rich Repeat (LRR) motifs, or both. Little is known, however, about the evolutionary events leading to the differential incorporation of LRR domains into Ig-containing tyrosine kinase receptors. Moreover, although FGFRs have been identified in many vertebrate species, few reports describe their existence in invertebrates. Information about the biological relevance of invertebrate FGFRs and evolutionary divergences between them and their vertebrate counterparts is therefore limited. Here, we characterized ApLRRTK, a neuronal cell-surface protein recently identified in Aplysia. We unveiled ApLRRTK as the first member of the FGFRs family deprived of Ig-like domains that instead contains extracellular LRR domains. We describe that ApLRRTK exhibits properties typical of canonical vertebrate FGFRs, including promotion of FGF activity, enhancement of neuritic outgrowth and signaling via MAPK and the transcription factor CREB. ApLRRTK also enhanced the synaptic efficiency of neurons known to mediate in vivo memory-related defensive behaviors. These data reveal a novel molecular regulator of neuronal function in invertebrates, provide the first evolutionary linkage between LRR proteins and FGFRs and unveil an unprecedented mechanism of FGFR gene diversification in primeval central nervous systems.

  2. Neurite, a finite difference large scale parallel program for the simulation of electrical signal propagation in neurites under mechanical loading.

    PubMed

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

    2015-01-01

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

  3. Regulation of neurite growth in immortalized mouse hypothalamic neurons and rat hippocampal primary cultures by teneurin C-terminal-associated peptide-1.

    PubMed

    Al Chawaf, A; St Amant, K; Belsham, D; Lovejoy, D A

    2007-02-23

    Teneurins are a highly conserved family of four type II transmembrane proteins that are expressed in the CNS. The protein possesses several functional domains including a unique bioactive 40-41 amino acid sequence at the extracellular terminus. Synthetic versions of this teneurin C-terminal-associated peptide (TCAP) can modulate cyclic AMP accumulation, cell proliferation and teneurin mRNA levels in vitro. Furthermore, i.c.v. injections of TCAP-1 into rat brain induce major changes in acoustic startle response behavior 3 weeks after administration, suggesting that the peptide may act to alter interneuron communication via changes in neurite and axon outgrowth. Synthetic mouse/rat TCAP-1 was used to treat cultured immortalized mouse hypothalamic cells, to determine if TCAP-1 could directly regulate neurite and axon growth. TCAP-1-treated cells showed a significant increase in the length of neurites accompanied by a marked increase in beta-tubulin transcription and translation as determined by real-time PCR and Western blot analysis, respectively. Changes in alpha-actinin-4 transcription and beta-actin protein expression were also noted. Immunofluorescence confocal microscopy using beta-tubulin antiserum showed enhanced resolution of beta-tubulin cytoskeletal elements throughout the cell. In order to determine if the effects of TCAP-1 could be reproduced in primary neuronal cultures, primary cultures of E18 rat hippocampal cells were treated with 100 nM TCAP-1. The TCAP-1-treated hippocampal cultures showed a significant increase in both the number of cells, dendritic branching and the presence of large and fasciculated beta-tubulin immunoreactive axons. These data suggest that TCAP acts, in part, as a functional region of the teneurins to regulate neurite and axonal growth of neurons.

  4. Retrograde degeneration of neurite membrane structural integrity of nerve growth cones following in vitro exposure to mercury.

    PubMed

    Leong, C C; Syed, N I; Lorscheider, F L

    2001-03-26

    Inhalation of mercury vapor (Hg0) inhibits binding of GTP to rat brain tubulin, thereby inhibiting tubulin polymerization into microtubules. A similar molecular lesion has also been observed in 80% of brains from patients with Alzheimer disease (AD) compared to age-matched controls. However the precise site and mode of action of Hg ions remain illusive. Therefore, the present study examined whether Hg ions could affect membrane dynamics of neurite growth cone morphology and behavior. Since tubulin is a highly conserved cytoskeletal protein in both vertebrates and invertebrates, we hypothesized that growth cones from animal species could be highly susceptible to Hg ions. To test this possibility, the identified, large Pedal A (PeA) neurons from the central ring ganglia of the snail Lymnoea stagnalis were cultured for 48 h in 2 ml brain conditioned medium (CM). Following neurite outgrowth, metal chloride solution (2 microl) of Hg, Al, Pb, Cd, or Mn (10(-7) M) was pressure applied directly onto individual growth cones. Time-lapse images with inverted microscopy were acquired prior to, during, and after the metal ion exposure. We demonstrate that Hg ions markedly disrupted membrane structure and linear growth rates of imaged neurites in 77% of all nerve growth cones. When growth cones were stained with antibodies specific for both tubulin and actin, it was the tubulin/microtubule structure that disintegrated following Hg exposure. Moreover, some denuded neurites were also observed to form neurofibrillary aggregates. In contrast, growth cone exposure to other metal ions did not effect growth cone morphology, nor was their motility rate compromised. To determine the growth suppressive effects of Hg ions on neuronal sprouting, cells were cultured either in the presence or absence of Hg ions. We found that in the presence of Hg ions, neuronal somata failed to sprout, whereas other metalic ions did not effect growth patterns of cultured PeA cells. We conclude that this

  5. Origins of circulating endothelial cells and endothelial outgrowth from blood

    PubMed Central

    Lin, Yi; Weisdorf, Daniel J.; Solovey, Anna; Hebbel, Robert P.

    2000-01-01

    Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood, and endothelial outgrowth has been observed from cultures of blood. In this study we seek insight into the origins of CEC and endothelial outgrowth from cultures of blood. Fluorescence in situ hybridization analysis of blood samples from bone marrow transplant recipients who had received gender-mismatched transplants 5–20 months earlier showed that most CEC in fresh blood had recipient genotype. Endothelial outgrowth from the same blood samples after 9 days in culture (5-fold expansion) was still predominantly of the recipient genotype. In contrast, endothelial outgrowth after ∼1 month (102-fold expansion) was mostly of donor genotype. Thus, recipient-genotype endothelial cells expanded only ∼20-fold over this period, whereas donor-genotype endothelial cells expanded ∼1000-fold. These data suggest that most CEC in fresh blood originate from vessel walls and have limited growth capability. Conversely, the data indicate that outgrowth of endothelial cells from cultures of blood is mostly derived from transplantable marrow-derived cells. Because these cells have more delayed outgrowth but a greater proliferative rate, our data suggest that they are derived from circulating angioblasts. PMID:10619863

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

    PubMed Central

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

    2015-01-01

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

  7. Temporal relation between neural activity and neurite pruning on a numerical model and a microchannel device with micro electrode array.

    PubMed

    Kondo, Yohei; Yada, Yuichiro; Haga, Tatsuya; Takayama, Yuzo; Isomura, Takuya; Jimbo, Yasuhiko; Fukayama, Osamu; Hoshino, Takayuki; Mabuchi, Kunihiko

    2017-04-29

    Synapse elimination and neurite pruning are essential processes for the formation of neuronal circuits. These regressive events depend on neural activity and occur in the early postnatal days known as the critical period, but what makes this temporal specificity is not well understood. One possibility is that the neural activities during the developmentally regulated shift of action of GABA inhibitory transmission lead to the critical period. Moreover, it has been reported that the shifting action of the inhibitory transmission on immature neurons overlaps with synapse elimination and neurite pruning and that increased inhibitory transmission by drug treatment could induce temporal shift of the critical period. However, the relationship among these phenomena remains unclear because it is difficult to experimentally show how the developmental shift of inhibitory transmission influences neural activities and whether the activities promote synapse elimination and neurite pruning. In this study, we modeled synapse elimination in neuronal circuits using the modified Izhikevich's model with functional shifting of GABAergic transmission. The simulation results show that synaptic pruning within a specified period like the critical period is spontaneously generated as a function of the developmentally shifting inhibitory transmission and that the specific firing rate and increasing synchronization of neural circuits are seen at the initial stage of the critical period. This temporal relationship was experimentally supported by an in vitro primary culture of rat cortical neurons in a microchannel on a multi-electrode array (MEA). The firing rate decreased remarkably between the 18-25 days in vitro (DIV), and following these changes in the firing rate, the neurite density was slightly reduced. Our simulation and experimental results suggest that decreasing neural activity due to developing inhibitory synaptic transmission could induce synapse elimination and neurite pruning

  8. Fine Needle Aspiration Cytology in Diagnosis of Pure Neuritic Leprosy

    PubMed Central

    Kumar, Bipin; Pradhan, Anju

    2011-01-01

    Leprosy is a chronic infection affecting mainly the skin and peripheral nerve. Pure neuritic form of this disease manifests by involvement of the nerve in the absence of skin lesions. Therefore, it can sometimes create a diagnostic problem. It often requires a nerve biopsy for diagnosis, which is an invasive procedure and may lead to neural deficit. Fine needle aspiration cytology (FNAC) of an affected nerve can be a valuable and less invasive procedure for the diagnosis of such cases. We report five suspected cases of pure neuritic Hansen's disease involving the common and superficial peroneal, ulnar, and median nerve, who underwent FNAC. Smears revealed nerve fibers infiltrated by chronic inflammatory cells in all cases, presence of epithelioid cells granulomas, and Langhans giant cells in three cases, and acid fast bacilli in two cases. In conclusion, FNAC is a safe, less invasive, and time saving procedure for the diagnosis of pure neuritic leprosy. PMID:21660285

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

    PubMed

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

    2014-01-01

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

  10. Neuroprotective Copper Bis(thiosemicarbazonato) Complexes Promote Neurite Elongation

    PubMed Central

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

    2014-01-01

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

  11. Material Stiffness Effects on Neurite Alignment to Photopolymerized Micropatterns

    PubMed Central

    2015-01-01

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

  12. Arf6 Guanine Nucleotide Exchange Factor Cytohesin-2 Binds to CCDC120 and Is Transported Along Neurites to Mediate Neurite Growth*

    PubMed Central

    Torii, Tomohiro; Miyamoto, Yuki; Tago, Kenji; Sango, Kazunori; Nakamura, Kazuaki; Sanbe, Atsushi; Tanoue, Akito; Yamauchi, Junji

    2014-01-01

    The mechanism of neurite growth is complicated, involving continuous cytoskeletal rearrangement and vesicular trafficking. Cytohesin-2 is a guanine nucleotide exchange factor for Arf6, an Arf family molecular switch protein, controlling cell morphological changes such as neuritogenesis. Here, we show that cytohesin-2 binds to a protein with a previously unknown function, CCDC120, which contains three coiled-coil domains, and is transported along neurites in differentiating N1E-115 cells. Transfection of the small interfering RNA (siRNA) specific for CCDC120 into cells inhibits neurite growth and Arf6 activation. When neurites start to extend, vesicles containing CCDC120 and cytohesin-2 are transported in an anterograde manner rather than a retrograde one. As neurites continue extension, anterograde vesicle transport decreases. CCDC120 knockdown inhibits cytohesin-2 localization into vesicles containing CCDC120 and diffuses cytohesin-2 in cytoplasmic regions, illustrating that CCDC120 determines cytohesin-2 localization in growing neurites. Reintroduction of the wild type CCDC120 construct into cells transfected with CCDC120 siRNA reverses blunted neurite growth and Arf6 activity, whereas the cytohesin-2-binding CC1 region-deficient CCDC120 construct does not. Thus, cytohesin-2 is transported along neurites by vesicles containing CCDC120, and it mediates neurite growth. These results suggest a mechanism by which guanine nucleotide exchange factor for Arf6 is transported to mediate neurite growth. PMID:25326380

  13. Chlorpyrifos- and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2.

    PubMed

    Sindi, Ramya A; Harris, Wayne; Arnott, Gordon; Flaskos, John; Lloyd Mills, Chris; Hargreaves, Alan J

    2016-10-01

    Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20h before exposure to CPF or CPO for 2-8h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length of axon-like processes compared to the control, respectively, retraction of neurites being observed within 2h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2h exposure and reduced levels of reactivity of the same antibody following 8h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation.

  14. Wisp2/CCN5 up-regulated in the central nervous system of GM3-only mice facilitates neurite formation in Neuro2a cells via integrin-Akt signaling

    SciTech Connect

    Ohkawa, Yuki; Ohmi, Yuhsuke; Tajima, Orie; Yamauchi, Yoshio; Furukawa, Keiko; Furukawa, Koichi

    2011-08-05

    Highlights: {yields} Wisp2/CCN5 was up-regulated in nervous tissues of GM3-only mutant mice. {yields} Wisp2/CCN5 was found in neurons more strongly in the mutant mice. {yields} Wisp2/CCN5 induces Akt phosphorylation via integrins and facilitates neurite formation. {yields} Wisp2/CCN5 conferred resistance to H{sub 2}O{sub 2}-induced apoptosis. {yields} Up-regulation of Wisp2/CCN5 in GM3-only mice seemed for protection of brains from neurodegeneration. -- Abstract: Wisp2/CCN5 belongs to CCN family proteins which are involved in cell proliferation, angiogenesis, tumorigenesis and wound healing. Although a number of studies on the roles of Wisp2/CCN5 in cancers have been reported, no study on the expression and function of Wisp2/CCN5 in the central nervous system has been reported. In this study, we focused on Wisp2/CCN5 that was up-regulated in nervous tissues in GM3-only mice. Over-expression of Wisp2/CCN5 enhanced neurite outgrowth potently after serum withdrawal with increased phosphorylation levels of Akt and ERKs. When cells were cultured with recombinant Wisp2/CCN5 proteins, more and longer neurites were formed than in the controls. Thus, we demonstrated for the first time that Wisp2/CCN5 facilitates neurite formation in a mouse neuroblastoma cell line, Neuro2a. Akt phosphorylation induced by recombinant Wisp2/CCN5 was suppressed after knockdown of integrin {beta}1. Moreover, Wisp2/CCN5-over-expressing cells were resistant to apoptosis induced by H{sub 2}O{sub 2}. These results suggested that secreted Wisp2/CCN5 induces Akt and ERK phosphorylation via integrins, and consequently facilitates neurite formation and conferred resistance to apoptosis. Up-regulation of Wisp2/CCN5 in GM3-only mice should be, therefore, a reaction to protect nervous tissues from neurodegeneration caused by ganglioside deficiency.

  15. Conditioned medium of dental pulp cells stimulated by Chinese propolis show neuroprotection and neurite extension in vitro.

    PubMed

    Kudo, Daichi; Inden, Masatoshi; Sekine, Shin-Ichiro; Tamaoki, Naritaka; Iida, Kazuki; Naito, Eiji; Watanabe, Kazuhiro; Kamishina, Hiroaki; Shibata, Toshiyuki; Hozumi, Isao

    2015-03-04

    The purpose of this study was to clarify the effect of Chinese propolis on the expression level of neurotrophic factors in dental pulp cells (DPCs). We also investigated that the effects of the conditioned medium (CM) of DPCs stimulated by the propolis against oxidative and endoplasmic reticulum (ER) stresses in human neuroblastoma SH-SY5Y cells, and on neurite extensions in rat adrenal pheochromocytoma PC12 cells. To investigate the effect of the propolis on the levels of neurotrophic factors in DPCs, we performed a qRT-PCR experiment. As results, NGF, but not BDNF and NT-3, in DPCs was significantly elevated by the propolis in a concentration-dependent manner. H2O2-induced cell death was significantly inhibited by the treatment with the CM of DPCs. In addition, the treatment with the propolis-stimulated CM of DPCs had a more protective effect than that with the CM of DPCs. We also examine the effect of the propolis-stimulated CM of DPCs against a tunicamycin-induced ER stress. The treatment with the propolis-stimulated CM as well as the CM of DPCs significantly inhibited tunicamycin-induced cell death. Moreover, the treatment with the propolis-stimulated CM of DPCs significantly induced neurite outgrowth from PC12 cells than that with the CM of DPCs. These results suggest that the CM of DPCs as well as DPCs will be an efficient source of new treatments for neurodegenerative diseases and that the propolis promote the advantage of the CM of DPCs via producing neurotrophic factors.

  16. Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain.

    PubMed

    Civiero, Laura; Cirnaru, Maria Daniela; Beilina, Alexandra; Rodella, Umberto; Russo, Isabella; Belluzzi, Elisa; Lobbestael, Evy; Reyniers, Lauran; Hondhamuni, Geshanthi; Lewis, Patrick A; Van den Haute, Chris; Baekelandt, Veerle; Bandopadhyay, Rina; Bubacco, Luigi; Piccoli, Giovanni; Cookson, Mark R; Taymans, Jean-Marc; Greggio, Elisa

    2015-12-01

    Leucine-rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21-activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21-activated kinases are serine-threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post-mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock-out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2-mediated pathophysiology. We propose p21-activated kinase 6 (PAK6) as a novel interactor of leucine-rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2-linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6.

  17. NGF-conjugated iron oxide nanoparticles promote differentiation and outgrowth of PC12 cells

    NASA Astrophysics Data System (ADS)

    Marcus, M.; Skaat, H.; Alon, N.; Margel, S.; Shefi, O.

    2014-12-01

    The search for regenerative agents that promote neuronal differentiation and repair is of great importance. Nerve growth factor (NGF) which is an essential contributor to neuronal differentiation has shown high pharmacological potential for the treatment of central neurodegenerative diseases such as Alzheimer's and Parkinson's. However, growth factors undergo rapid degradation, leading to a short biological half-life. In our study, we describe a new nano-based approach to enhance the NGF activity resulting in promoted neuronal differentiation. We covalently conjugated NGF to iron oxide nanoparticles (NGF-NPs) and studied the effect of the novel complex on the differentiation of PC12 cells. We found that the NGF-NP treatment, at the same concentration as free NGF, significantly promoted neurite outgrowth and increased the complexity of the neuronal branching trees. Examination of neuronal differentiation gene markers demonstrated higher levels of expression in PC12 cells treated with the conjugated factor. By manipulating the NGF specific receptor, TrkA, we have demonstrated that NGF-NPs induce cell differentiation via the regular pathway. Importantly, we have shown that NGF-NPs undergo slower degradation than free NGF, extending their half-life and increasing NGF availability. Even a low concentration of conjugated NGF treatment has led to an effective response. We propose the use of the NGF-NP complex which has magnetic characteristics, also as a useful method to enhance NGF efficiency and activity, thus, paving the way for substantial neuronal repair therapeutics.The search for regenerative agents that promote neuronal differentiation and repair is of great importance. Nerve growth factor (NGF) which is an essential contributor to neuronal differentiation has shown high pharmacological potential for the treatment of central neurodegenerative diseases such as Alzheimer's and Parkinson's. However, growth factors undergo rapid degradation, leading to a short

  18. Neurite guidance and three-dimensional confinement via compliant semiconductor scaffolds.

    PubMed

    Cavallo, Francesca; Huang, Yu; Dent, Erik W; Williams, Justin C; Lagally, Max G

    2014-12-23

    Neurons are often cultured in vitro on a flat, open, and rigid substrate, a platform that does not reflect well the native microenvironment of the brain. To address this concern, we have developed a culturing platform containing arrays of microchannels, formed in a crystalline-silicon nanomembrane (NM) resting on polydimethylsiloxane; this platform will additionally enable active sensing and stimulation at the local scale, via devices fabricated in the silicon. The mechanical properties of the composite Si/compliant substrate nanomaterial approximate those of neural tissue. The microchannels, created in the NM by strain engineering, demonstrate strong guidance of neurite outgrowth. Using plasma techniques, we developed a means to coat just the inside surface of these channels with an adhesion promoter (poly-d-lysine). For NM channels with openings larger than the cross-sectional area of a single axon, strong physical confinement and guidance of axons through the channels are observed. Imaging of axons that grow in channels with openings that approximate the size of an axon suggests that a tight seal exists between the cell membrane and the inner surface of the channel, mimicking a myelin sheath. Such a tight seal of the cell membrane with the channel surface would make this platform an attractive candidate for future neuronal repair. Results of measurements of impedance and photoluminescence of bare NM channels are comparable to those on a flat NM, demonstrating electrical and optical modalities of our platform and suggesting that this scaffold can be expanded for active sensing and monitoring of neuron cellular processes in conditions in which they exist naturally.

  19. Intracellular signaling is changed after clustering of the neural cell adhesion molecules axonin-1 and NgCAM during neurite fasciculation

    PubMed Central

    1996-01-01

    Neural cell adhesion molecules of the immunoglobulin/fibronectin type III family on axons have been implicated in promotion of neurite outgrowth, fasciculation, and the mediation of specific cell adhesion. The present study demonstrates that two of these molecules on dorsal root ganglion neurons are associated with distinct protein kinases, axonin-1 with the src-related nonreceptor tyrosine kinase fyn and NgCAM with a casein kinase II-related activity and a serine/ threonine kinase related to S6 kinase. When neurites grew without contacts involving axonin-1 and NgCAM, strong fyn kinase activity was associated with axonin-1, whereas the NgCAM-associated kinase activities were low. Clustering of axonin-1 with NgCAM induced by the formation of cell-cell contacts correlated with a reduction of the axonin-1-associated fyn activity and an increased phosphorylation of NgCAM by the associated casein kinase II-related activity. Thus, axonin-1 and NgCAM trigger distinctive intracellular signals during in vitro differentiation depending on their state of association. PMID:8858178

  20. Inhibition of N1-Src kinase by a specific SH3 peptide ligand reveals a role for N1-Src in neurite elongation by L1-CAM

    PubMed Central

    Keenan, Sarah; Wetherill, Sarah J.; Ugbode, Christopher I.; Chawla, Sangeeta; Brackenbury, William J.; Evans, Gareth J. O.

    2017-01-01

    In the mammalian brain the ubiquitous tyrosine kinase, C-Src, undergoes splicing to insert short sequences in the SH3 domain to yield N1- and N2-Src. We and others have previously shown that the N-Srcs have altered substrate specificity and kinase activity compared to C-Src. However, the exact functions of the N-Srcs are unknown and it is likely that N-Src signalling events have been misattributed to C-Src because they cannot be distinguished by conventional Src inhibitors that target the kinase domain. By screening a peptide phage display library, we discovered a novel ligand (PDN1) that targets the unique SH3 domain of N1-Src and inhibits N1-Src in cells. In cultured neurons, PDN1 fused to a fluorescent protein inhibited neurite outgrowth, an effect that was mimicked by shRNA targeting the N1-Src microexon. PDN1 also inhibited L1-CAM-dependent neurite elongation in cerebellar granule neurons, a pathway previously shown to be disrupted in Src−/− mice. PDN1 therefore represents a novel tool for distinguishing the functions of N1-Src and C-Src in neurons and is a starting point for the development of a small molecule inhibitor of N1-Src. PMID:28220894

  1. Reactive oxygen species induce neurite degeneration before induction of cell death

    PubMed Central

    Fukui, Koji

    2016-01-01

    Reactive oxygen species (ROS) induce neuronal cell death in a time- and concentration-dependent manner. Treatment of cultured cells with a low concentration of hydrogen peroxide induces neurite degeneration, but not cell death. Neurites (axons and dendrites) are vulnerable to ROS. Neurite degeneration (shrinkage, accumulation, and fragmentation) has been found in neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. However, the mechanism of ROS-related neurite degeneration is not fully understood. Many studies have demonstrated the relationship between mitochondrial dysfunction and microtubule destabilization. These dysfunctions are deeply related to changes in calcium homeostasis and ROS production in neurites. Treatment with antioxidant substances, such as vitamin E, prevents neurite degeneration in cultured cells. This review describes the possibility that ROS induces neurite degeneration before the induction of cell death. PMID:27895381

  2. Hierarchical Disabled-1 Tyrosine Phosphorylation in Src family Kinase Activation and Neurite Formation

    PubMed Central

    Katyal, Sachin; Gao, Zhihua; Monckton, Elizabeth; Glubrecht, Darryl; Godbout, Roseline

    2013-01-01

    There are two developmentally regulated alternatively spliced forms of Disabled-1 (Dab1) in the chick retina: an early form (Dab1-E) expressed in retinal precursor cells and a late form (Dab1-L) expressed in neuronal cells. The main difference between these two isoforms is the absence of two Src family kinase (SFK) recognition sites in Dab1-E. Both forms retain two Abl/Crk/Nck recognition sites implicated in the recruitment of SH2 domain-containing signaling proteins. One of the Dab1-L-specific SFK recognition sites, at tyrosine(Y)-198, has been shown to be phosphorylated in Reelin-stimulated neurons. Here, we use Reelin-expressing primary retinal cultures to investigate the role of the four Dab1 tyrosine phosphorylation sites on overall tyrosine phosphorylation, Dab1 phosphorylation, SFK activation and neurite formation. We show that Y198 is essential but not sufficient for maximal Dab1 phosphorylation, SFK activation and neurite formation, with Y232 and Y220 playing particularly important roles in SFK activation and neuritogenesis, and Y185 having modifying effects secondary to Y232 and Y220. Our data support a role for all four Dab1 tyrosine phosphorylation sites in mediating the spectrum of activities associated with Reelin-Dab1 signaling in neurons. PMID:17350651

  3. Fibroblast growth factor 2 promotes primitive endoderm development in bovine blastocyst outgrowths.

    PubMed

    Yang, Qi En; Fields, Sarah D; Zhang, Kun; Ozawa, Manabu; Johnson, Sally E; Ealy, Alan D

    2011-11-01

    Primitive endoderm (PE) is the second extraembryonic tissue to form during embryogenesis in mammals. The PE develops from pluripotent cells of the blastocyst inner cell mass. Experimental results described herein provide evidence that FGF2 stimulates PE development during bovine blastocyst development in vitro. Bovine blastocysts were cultured individually on a feeder layer-free, Matrigel-coated surface in the presence or absence of FGF2. A majority of blastocysts cultures formed outgrowths (76.8%) and the rate of outgrowth formation was not affected by FGF2 supplementation. However, supplementation with FGF2 increased the incidence of PE outgrowths on Days 13 and 15 after in vitro fertilization. Presumptive PE cultures contained cells with a phenotype distinct from trophectoderm (TE). Cell identity was validated by expression of GATA4 and GATA6 mRNA and transferrin protein, all markers of the PE lineage. Expression of GATA4 occurred coincident with blastocyst expansion and hatching. These cells did not express IFNT and CDX2 (TE lineage markers). Profiles of FGF receptor (FGFR) isoforms were distinct between PE and TE cultures. Specifically, FGFR1b and FGFR1c were the predominant FGFR transcripts in PE whereas FGFR2b transcripts were abundant in TE. Supplementation with FGF2 increased the mitotic index of PE but not TE. Moreover, FGF signaling appears important for initiation of PE formation in blastocysts, presumably by lineage committal from NANOG-positive epiblast cells, because chemical disruption of FGFR kinase activity with PD173074 reduces GATA4 expression and increases NANOG expression. Collectively, these results indicate that FGF2 and potentially other FGFs specify PE formation and mediate PE proliferation during early pregnancy in cattle.

  4. Fine-tuned SRF activity controls asymmetrical neuronal outgrowth: implications for cortical migration, neural tissue lamination and circuit assembly

    PubMed Central

    Scandaglia, Marilyn; Benito, Eva; Morenilla-Palao, Cruz; Fiorenza, Anna; del Blanco, Beatriz; Coca, Yaiza; Herrera, Eloísa; Barco, Angel

    2015-01-01

    The stimulus-regulated transcription factor Serum Response Factor (SRF) plays an important role in diverse neurodevelopmental processes related to structural plasticity and motile functions, although its precise mechanism of action has not yet been established. To further define the role of SRF in neural development and distinguish between cell-autonomous and non cell-autonomous effects, we bidirectionally manipulated SRF activity through gene transduction assays that allow the visualization of individual neurons and their comparison with neighboring control cells. In vitro assays showed that SRF promotes survival and filopodia formation and is required for normal asymmetric neurite outgrowth, indicating that its activation favors dendrite enlargement versus branching. In turn, in vivo experiments demonstrated that SRF-dependent regulation of neuronal morphology has important consequences in the developing cortex and retina, affecting neuronal migration, dendritic and axonal arborization and cell positioning in these laminated tissues. Overall, our results show that the controlled and timely activation of SRF is essential for the coordinated growth of neuronal processes, suggesting that this event regulates the switch between neuronal growth and branching during developmental processes. PMID:26638868

  5. Studies of Schwann cell proliferation. III. Evidence for the surface localization of the neurite mitogen

    PubMed Central

    1980-01-01

    In the preceding paper (Salzer et al., 1980, J. Cell Biol. 84:753-- 766), evidence was presented that a neurite membrane fraction could be used to stimulate Schwann cell proliferation in culture. In this study, we present evidence that the mitogenic signal by which intact neurites or neurite membranes stimulate Schwann cell proliferation is located at the neurite surface. This conclusion is based on the following observations: (a) stimulation of Schwann cell proliferation by neurons requires direct contact between neurites and Schwann cells, separation of the two cells by a permeable collagen diaphragm 6 microns thick prevents Schwann cell proliferation; (b) treatment of intact neurites with trypsin before preparation of neurite membranes abolishes the ability of these membranes to be mitogenic for Schwann cells; and (c) the mitogenic activity of neurite homogenates is exclusively localized in the particulate rather than the soluble fraction of the homogenate. The mitogenic component on the neurite surface is heat labile, and is inactivated by aldehyde fixation. Preliminary data suggest that the mitogenic effect of neurite on Schwann cells is not mediated by 3',5'- cyclic AMP. PMID:6153659

  6. Can hippocampal neurites and growth cones climb over obstacles?

    PubMed

    Lien, Thuy Linh; Ban, Jelena; Tormen, Massimo; Migliorini, Elisa; Grenci, Gianluca; Pozzato, Alessandro; Torre, Vincent

    2013-01-01

    Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 µm wide, with 4 µm period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 - do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles.

  7. The Rif GTPase regulates cytoskeletal signaling from plexinA4 to promote neurite retraction.

    PubMed

    Fan, Lifei; Yan, Huijuan; Pellegrin, Stephanie; Morigen; Mellor, Harry

    2015-03-17

    The small GTPase Rif is required for the early stages of dendritic spine formation in neurons, acting through the formin mDia2 to control actin polymerization. Rif is expressed at high levels in the brain, suggesting broader roles in neuronal function. We screened a yeast two-hybrid cDNA library to identify additional binding partners for Rif of potential relevance to neuronal function. We found that Rif interacts with FARP1, a neuronal activator of the RhoA GTPase. We show that Rif has two separate roles in FARP1 regulation-in controlling its association with plexinA4, and in releasing active RhoA from a plexinA4/FARP1 complex. The regulation of FARP1 by Rif promotes neurite retraction in cells stimulated with the semaphorin Sema6A.

  8. Light Signaling in Bud Outgrowth and Branching in Plants

    PubMed Central

    Leduc, Nathalie; Roman, Hanaé; Barbier, François; Péron, Thomas; Huché-Thélier, Lydie; Lothier, Jérémy; Demotes-Mainard, Sabine; Sakr, Soulaiman

    2014-01-01

    Branching determines the final shape of plants, which influences adaptation, survival and the visual quality of many species. It is an intricate process that includes bud outgrowth and shoot extension, and these in turn respond to environmental cues and light conditions. Light is a powerful environmental factor that impacts multiple processes throughout plant life. The molecular basis of the perception and transduction of the light signal within buds is poorly understood and undoubtedly requires to be further unravelled. This review is based on current knowledge on bud outgrowth-related mechanisms and light-mediated regulation of many physiological processes. It provides an extensive, though not exhaustive, overview of the findings related to this field. In parallel, it points to issues to be addressed in the near future. PMID:27135502

  9. Inhibition of Bacillus anthracis Spore Outgrowth by Nisin▿

    PubMed Central

    Gut, Ian M.; Prouty, Angela M.; Ballard, Jimmy D.; van der Donk, Wilfred A.; Blanke, Steven R.

    2008-01-01

    The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several Bacillus species. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. By using B. anthracis Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC50) and an IC90 of 0.57 μM and 0.90 μM, respectively. Viable B. anthracis organisms were not recoverable from cultures containing concentrations of nisin greater than the IC90. These studies demonstrated that spores lose heat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism of inhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for the action of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing the establishment of oxidative metabolism and the membrane potential in germinating spores. On the other hand, nisin had no detectable effects on the typical changes associated with the dissolution of the outer spore structures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncoupling of two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism and the shedding of the external spore structures. PMID:18809941

  10. IPP5 inhibits neurite growth in primary sensory neurons by maintaining TGF-β/Smad signaling.

    PubMed

    Han, Qing-Jian; Gao, Nan-Nan; Guo-QiangMa; Zhang, Zhen-Ning; Yu, Wen-Hui; Pan, Jing; Wang, Qiong; Zhang, Xu; Bao, Lan

    2013-01-15

    During nerve regeneration, neurite growth is regulated by both intrinsic molecules and extracellular factors. Here, we found that inhibitor 5 of protein phosphatase 1 (IPP5), a newly identified inhibitory subunit of protein phosphatase 1 (PP1), inhibited neurite growth in primary sensory neurons as an intrinsic regulator. IPP5 was highly expressed in the primary sensory neurons of rat dorsal root ganglion (DRG) and was downregulated after sciatic nerve axotomy. Knocking down IPP5 with specific shRNA increased the length of the longest neurite, the total neurite length and the number of neurite ends in cultured rat DRG neurons. Mutation of the PP1-docking motif K(8)IQF(11) or the PP1-inhibiting motif at Thr(34) eliminated the IPP5-induced inhibition of neurite growth. Furthermore, biochemical experiments showed that IPP5 interacted with type I transforming growth factor-β receptor (TβRI) and PP1 and enhanced transforming growth factor-β (TGF-β)/Smad signaling in a PP1-dependent manner. Overexpressing IPP5 in DRG neurons aggravated TGF-β-induced inhibition of neurite growth, which was abolished by blocking PP1 or IPP5 binding to PP1. Blockage of TGF-β signaling with the TβRI inhibitor SB431542 or Smad2 shRNA attenuated the IPP5-induced inhibition of neurite growth. Thus, these data indicate that selectively expressed IPP5 inhibits neurite growth by maintaining TGF-β signaling in primary sensory neurons.

  11. Growth cone-like waves transport actin and promote axonogenesis and neurite branching

    PubMed Central

    Flynn, Kevin C.; Pak, Chi W.; Shaw, Alisa E.; Bradke, Frank; Bamburg, James R.

    2010-01-01

    Axonogenesis involves a shift from uniform delivery of materials to all neurites to preferential delivery to the putative axon, supporting its more rapid extension. Waves, growth cone-like structures that propagate down the length of neurites, were shown previously to correlate with neurite growth in dissociated cultured hippocampal neurons. Waves are similar to growth cones in their structure, composition and dynamics. Here, we report that waves form in all undifferentiated neurites, but occur more frequently in the future axon during initial neuronal polarization. Moreover, wave frequency and their impact on neurite growth are altered in neurons treated with stimuli that enhance axonogenesis. Coincident with wave arrival, growth cones enlarge and undergo a marked increase in dynamics. Through their engorgement of filopodia along the neurite shaft, waves can induce de novo neurite branching. Actin in waves maintains much of its cohesiveness during transport whereas actin in non-wave regions of the neurite rapidly diffuses as measured by live cell imaging of photoactivated GFP-actin and photoconversion of Dendra-actin. Thus, waves represent an alternative axonal transport mechanism for actin. Waves also occur in neurons in organotypic hippocampal slices where they propagate along neurites in the dentate gyrus and the CA regions and induce branching. Taken together, our results indicate that waves are physiologically relevant and contribute to axon growth and branching via the transport of actin and by increasing growth cone dynamics. PMID:19513994

  12. Organic and inorganic lead inhibit neurite growth in vertebrate and invertebrate neurons in culture.

    PubMed

    Audesirk, G; Shugarts, D; Nelson, G; Przekwas, J

    1989-12-01

    Neurons from brains of chick embryos and pond snails (Lymnaea stagnalis) were cultured for 3 to 4 d in the presence of no toxins, inorganic lead (PbCl2), or organic lead (triethyl lead chloride). In chick neurons, inorganic lead reduced the percentage of cells that grew neurites (IC50 = 270 microM total lead, approximately 70 nM free Pb2+) but did not reduce the number of neurites per cell or the mean neurite length. Triethyl lead reduced the percentage of cells that grew neurites (IC50 = 0.24 microM) and the mean neurite length (extrapolated IC50 = 3.6 microM) but did not reduce the number of neurites per cell. In Lymnaea neurons, inorganic lead reduced the percentage of cells that grew neurites (IC50 = 13 microM total lead; approximately 10 nM free Pb2+). Triethyl lead reduced the percentage of cells that grew neurites (IC50 = 0.4 microM) and exerted significant toxicity at 0.2 microM. The two forms of lead affected neurite growth in qualitatively different ways, which suggests that their mechanisms of action are different.

  13. Quercetin promotes neurite growth through enhancing intracellular cAMP level and GAP-43 expression.

    PubMed

    Chen, Ming-Ming; Yin, Zhi-Qi; Zhang, Lu-Yong; Liao, Hong

    2015-09-01

    The present study was designed to investigate the role of quercetin on neurite growth in N1E-115 cells and the underlying mechanisms. Quercetin was evaluated for its effects on cell numbers of neurites, neurite length, intracellular cAMP content, and Gap-43 expression in N1E-115 cells in vitro by use of microscopy, LANCE(tm) cAMP 384 kit, and Western blot analysis, respectively. Our results showed that quercetin could increase the neurite length in a concentration-dependent manner, but had no effect on the numbers of cells. Quercetin significantly increased the expression of cellular cAMP in a time- and concentration-dependent manner. The Gap-43 expression was up-regulated in a time-dependent manner. In conclusion, quercetin could promote neurite growth through increasing the intracellular cAMP level and Gap-43 expression.

  14. The X-Linked Autism Protein KIAA2022/KIDLIA Regulates Neurite Outgrowth via N-Cadherin and δ-Catenin Signaling

    PubMed Central

    2016-01-01

    Abstract Our previous work showed that loss of the KIAA2022 gene protein results in intellectual disability with language impairment and autistic behavior (KIDLIA, also referred to as XPN). However, the cellular and molecular alterations resulting from a loss of function of KIDLIA and its role in autism with severe intellectual disability remain unknown. Here, we show that KIDLIA plays a key role in neuron migration and morphogenesis. We found that KIDLIA is distributed exclusively in the nucleus. In the developing rat brain, it is expressed only in the cortical plate and subplate region but not in the intermediate or ventricular zone. Using in utero electroporation, we found that short hairpin RNA (shRNA)-mediated knockdown of KIDLIA leads to altered neuron migration and a reduction in dendritic growth and disorganized apical dendrite projections in layer II/III mouse cortical neurons. Consistent with this, in cultured rat neurons, a loss of KIDLIA expression also leads to suppression of dendritic growth and branching. At the molecular level, we found that KIDLIA suppression leads to an increase in cell-surface N-cadherin and an elevated association of N-cadherin with δ-catenin, resulting in depletion of free δ-catenin in the cytosolic compartment. The reduced availability of cytosolic δ-catenin leads to elevated RhoA activity and reduced actin dynamics at the dendritic growth cone. Furthermore, in neurons with KIDLIA knockdown, overexpression of δ-catenin or inhibition of RhoA rescues actin dynamics, dendritic growth, and branching. These findings provide the first evidence on the role of the novel protein KIDLIA in neurodevelopment and autism with severe intellectual disability. PMID:27822498

  15. The X-Linked Autism Protein KIAA2022/KIDLIA Regulates Neurite Outgrowth via N-Cadherin and δ-Catenin Signaling.

    PubMed

    Gilbert, James; Man, Heng-Ye

    2016-01-01

    Our previous work showed that loss of the KIAA2022 gene protein results in intellectual disability with language impairment and autistic behavior (KIDLIA, also referred to as XPN). However, the cellular and molecular alterations resulting from a loss of function of KIDLIA and its role in autism with severe intellectual disability remain unknown. Here, we show that KIDLIA plays a key role in neuron migration and morphogenesis. We found that KIDLIA is distributed exclusively in the nucleus. In the developing rat brain, it is expressed only in the cortical plate and subplate region but not in the intermediate or ventricular zone. Using in utero electroporation, we found that short hairpin RNA (shRNA)-mediated knockdown of KIDLIA leads to altered neuron migration and a reduction in dendritic growth and disorganized apical dendrite projections in layer II/III mouse cortical neurons. Consistent with this, in cultured rat neurons, a loss of KIDLIA expression also leads to suppression of dendritic growth and branching. At the molecular level, we found that KIDLIA suppression leads to an increase in cell-surface N-cadherin and an elevated association of N-cadherin with δ-catenin, resulting in depletion of free δ-catenin in the cytosolic compartment. The reduced availability of cytosolic δ-catenin leads to elevated RhoA activity and reduced actin dynamics at the dendritic growth cone. Furthermore, in neurons with KIDLIA knockdown, overexpression of δ-catenin or inhibition of RhoA rescues actin dynamics, dendritic growth, and branching. These findings provide the first evidence on the role of the novel protein KIDLIA in neurodevelopment and autism with severe intellectual disability.

  16. Comparison of Human Induced Pluripotent Stem Cell-Derived Neurons and Rat Primary CorticalNeurons as In Vitro Models of Neurite Outgrowth

    EPA Science Inventory

    High-throughput assays that can quantify chemical-induced changes at the cellular and molecular level have been recommended for use in chemical safety assessment. High-throughput, high content imaging assays for the key cellular events of neurodevelopment have been proposed to ra...

  17. The Neurite Outgrowth Inhibitory Nogo-A-Δ20 Region Is an Intrinsically Disordered Segment Harbouring Three Stretches with Helical Propensity

    PubMed Central

    Bibow, Stefan; Schwab, Martin E.; Riek, Roland

    2016-01-01

    Functional recovery from central neurotrauma, such as spinal cord injury, is limited by myelin-associated inhibitory proteins. The most prominent example, Nogo-A, imposes an inhibitory cue for nerve fibre growth via two independent domains: Nogo-A-Δ20 (residues 544–725 of the rat Nogo-A sequence) and Nogo-66 (residues 1026–1091). Inhibitory signalling from these domains causes a collapse of the neuronal growth cone via individual receptor complexes, centred around sphingosine 1-phosphate receptor 2 (S1PR2) for Nogo-A-Δ20 and Nogo receptor 1 (NgR1) for Nogo-66. Whereas the helical conformation of Nogo-66 has been studied extensively, only little structural information is available for the Nogo-A-Δ20 region. We used nuclear magnetic resonance (NMR) spectroscopy to assess potential residual structural propensities of the intrinsically disordered Nogo-A-Δ20. Using triple resonance experiments, we were able to assign 94% of the non-proline backbone residues. While secondary structure analysis and relaxation measurements highlighted the intrinsically disordered character of Nogo-A-Δ20, three stretches comprising residues 561EAIQESL567, 639EAMNVALKALGT650, and 693SNYSEIAK700 form transient α-helical structures. Interestingly, 561EAIQESL567 is situated directly adjacent to one of the most conserved regions of Nogo-A-Δ20 that contains a binding motif for β1-integrin. Likewise, 639EAMNVALKALGT650 partially overlaps with the epitope recognized by 11C7, a Nogo-A-neutralizing antibody that promotes functional recovery from spinal cord injury. Diffusion measurements by pulse-field gradient NMR spectroscopy suggest concentration- and oxidation state-dependent dimerisation of Nogo-A-Δ20. Surprisingly, NMR and isothermal titration calorimetry (ITC) data could not validate previously shown binding of extracellular loops of S1PR2 to Nogo-A-Δ20. PMID:27611089

  18. ACTIVATION OF ERK1 AND ERK2 IS REQUIRED FOR MANGANESE-INDUCED NEURITE OUTGROWTH IN RAT PHEOCHROMOCYTOMA (PC12) CELLS. (R826248)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  19. Recombinant DNA vaccine against inhibition of neurite outgrowth promotes functional recovery associated with endogeous NGF expression in spinal cord hemisected adult rats.

    PubMed

    Zhang, Yi; Hao, Chun-Guang; Hu, Li-Qun; Dong, Jian; Wei, Peng; Xu, Dan; Xiao, Zhi-Cheng; Wang, Ting-Hua

    2009-09-01

    Axonal regeneration across the site of spinal cord lesion is often aborted in adult mammalian species. The use of DNA vaccine to nullify the inhibitory molecules has been shown to be effective in promoting axonal regeneration in injured spinal cord. The possible molecular mechanisms, however, remain to be elucidated. The present study showed that the administration of recombinant DNA vaccine encoding multiple domains, Nogo-66, Nogo-N, TnR, and MAG, significantly improved hindlimb locomotor functions in rats subjected to ablation of the dorsal halves of the cord. Western blot analysis demonstrated that nerve growth factor (NGF) levels in the spinal cord of immunized rats were significantly upregulated than those of control rats. Immunohistochemistry as well as in situ hybridization confirmed that NGF was expressed in neurons of the spinal cord. These findings indicated that functional recovery in immunized rats could be correlated with endogeous NGF expression in hemisected rat spinal cords.

  20. A Small Molecule p75NTR Ligand, LM11A-31, Reverses Cholinergic Neurite Dystrophy in Alzheimer's Disease Mouse Models with Mid- to Late-Stage Disease Progression

    PubMed Central

    Simmons, Danielle A.; Knowles, Juliet K.; Belichenko, Nadia P.; Banerjee, Gargi; Finkle, Carly; Massa, Stephen M.; Longo, Frank M.

    2014-01-01

    Degeneration of basal forebrain cholinergic neurons contributes significantly to the cognitive deficits associated with Alzheimer's disease (AD) and has been attributed to aberrant signaling through the neurotrophin receptor p75 (p75NTR). Thus, modulating p75NTR signaling is considered a promising therapeutic strategy for AD. Accordingly, our laboratory has developed small molecule p75NTR ligands that increase survival signaling and inhibit amyloid-β-induced degenerative signaling in in vitro studies. Previous work found that a lead p75NTR ligand, LM11A-31, prevents degeneration of cholinergic neurites when given to an AD mouse model in the early stages of disease pathology. To extend its potential clinical applications, we sought to determine whether LM11A-31 could reverse cholinergic neurite atrophy when treatment begins in AD mouse models having mid- to late stages of pathology. Reversing pathology may have particular clinical relevance as most AD studies involve patients that are at an advanced pathological stage. In this study, LM11A-31 (50 or 75 mg/kg) was administered orally to two AD mouse models, Thy-1 hAPPLond/Swe (APPL/S) and Tg2576, at age ranges during which marked AD-like pathology manifests. In mid-stage male APPL/S mice, LM11A-31 administered for 3 months starting at 6–8 months of age prevented and/or reversed atrophy of basal forebrain cholinergic neurites and cortical dystrophic neurites. Importantly, a 1 month LM11A-31 treatment given to male APPL/S mice (12–13 months old) with late-stage pathology reversed the degeneration of cholinergic neurites in basal forebrain, ameliorated cortical dystrophic neurites, and normalized increased basal forebrain levels of p75NTR. Similar results were seen in female Tg2576 mice. These findings suggest that LM11A-31 can reduce and/or reverse fundamental AD pathologies in late-stage AD mice. Thus, targeting p75NTR is a promising approach to reducing AD-related degenerative processes that have progressed

  1. Gene expression profile of brain regions reflecting aberrations in nervous system development targeting the process of neurite extension of rat offspring exposed developmentally to glycidol.

    PubMed

    Akane, Hirotoshi; Saito, Fumiyo; Shiraki, Ayako; Imatanaka, Nobuya; Akahori, Yumi; Itahashi, Megu; Wang, Liyun; Shibutani, Makoto

    2014-12-01

    We previously found that exposure to glycidol at 1000 ppm in drinking water caused axonopathy in maternal rats and aberrations in late-stage hippocampal neurogenesis, targeting the process of neurite extension in offspring. To identify the profile of developmental neurotoxicity of glycidol, pregnant Sprague-Dawley rats were given drinking water containing glycidol from gestational day 6 until weaning on day 21 after delivery, and offspring at 0, 300 and 1000 ppm were subjected to region-specific global gene expression profiling. Four brain regions were selected to represent both cerebral and cerebellar tissues, i.e., the cingulate cortex, corpus callosum, hippocampal dentate gyrus and cerebellar vermis. Downregulated genes in the dentate gyrus were related to axonogenesis (Nfasc), myelination (Mal, Mrf and Ugt8), and cell proliferation (Aurkb and Ndc80) at ≥ 300 ppm, and upregulated genes were related to neural development (Frzb and Fzd6) at 1000 ppm. Upregulation was observed for genes related to myelination (Kl, Igf2 and Igfbp2) in the corpus callosum and axonogenesis and neuritogenesis (Efnb3, Tnc and Cd44) in the cingulate cortex, whereas downregulation was observed for genes related to synaptic transmission (Thbs2 and Ccl2) in the cerebellar vermis; all of these changes were mostly observed at 1000 ppm. Altered gene expression of Cntn3, which functions on neurite outgrowth-promotion, was observed in all four brain regions at 1000 ppm. Gene expression profiles suggest that developmental exposure to glycidol affected plasticity of neuronal networks in the broad brain areas, and dentate gyrus neurogenesis may be the sensitive target of this type of toxicity.

  2. gp130 cytokines are positive signals triggering changes in gene expression and axon outgrowth in peripheral neurons following injury

    PubMed Central

    Zigmond, Richard E.

    2012-01-01

    Adult peripheral neurons, in contrast to adult central neurons, are capable of regeneration after axonal damage. Much attention has focused on the changes that accompany this regeneration in two places, the distal nerve segment (where phagocytosis of axonal debris, changes in the surface properties of Schwann cells, and induction of growth factors and cytokines occur) and the neuronal cell body (where dramatic changes in cell morphology and gene expression occur). The changes in the axotomized cell body are often referred to as the “cell body response.” The focus of the current review is a family of cytokines, the glycoprotein 130 (gp130) cytokines, which produce their actions through a common gp130 signaling receptor and which function as injury signals for axotomized peripheral neurons, triggering changes in gene expression and in neurite outgrowth. These cytokines play important roles in the responses of sympathetic, sensory, and motor neurons to injury. The best studied of these cytokines in this context are leukemia inhibitory factor (LIF) and interleukin (IL)-6, but experiments with conditional gp130 knockout animals suggest that other members of this family, not yet determined, are also involved. The primary gp130 signaling pathway shown to be involved is the activation of Janus kinase (JAK) and the transcription factors Signal Transducers and Activators of Transcription (STAT), though other downstream pathways such as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) may also play a role. gp130 signaling may involve paracrine, retrograde, and autocrine actions of these cytokines. Recent studies suggest that manipulation of this cytokine system can also stimulate regeneration by injured central neurons. PMID:22319466

  3. Rabies virus neuritic paralysis: immunopathogenesis of nonfatal paralytic rabies.

    PubMed Central

    Weiland, F; Cox, J H; Meyer, S; Dahme, E; Reddehase, M J

    1992-01-01

    Two pathogenetically distinct disease manifestations are distinguished in a murine model of primary rabies virus infection with the Evelyn-Rokitnicky-Abelseth strain, rabies virus neuritic paralysis (RVNP) and fatal encephalopathogenic rabies. RVNP develops with high incidence in immunocompetent mice after intraplantar infection as a flaccid paralysis restricted to the infected limb. The histopathologic correlate of this monoplegia is a degeneration of the myelinated motor neurons of the peripheral nerve involved. While, in this model, fatal encephalopathogenic rabies develops only after depletion of the CD4 subset of T lymphocytes and without contribution of the CD8 subset, RVNP is identified as an immunopathological process in which both the CD4 and CD8 subsets of T lymphocytes are critically implicated. Images PMID:1629964

  4. Long-term neurite orientation on astrocyte monolayers aligned by microtopography.

    PubMed

    Sørensen, Annette; Alekseeva, Tijna; Katechia, Kashyap; Robertson, Mary; Riehle, Mathis O; Barnett, Susan C

    2007-12-01

    After spinal cord injury neuronal connections are not easily re-established. Success has been hampered by the lack of orientation of neurites inside scar tissue and a lack of neurites crossing out of the site of injury. Oriented scaffolds in biodegradable polymers could be an excellent way to support both the orientation of neurites within the injury site as well as aiding their crossing out of the lesion. To establish the validity of using grooved micro-topography in polycaprolactone in combination with glia we have studied the long-term (3 weeks) orientation of neuronal cells on monolayers of astrocytes on the top of grooved topographies of various dimensions. We find that neurites are significantly aligned by groove/ridge type topographies which are "buried" under a monolayer of astrocytes for up to 3 weeks. This alignment is significantly lower than that of neurites growing directly on the topography, but these neurons do not survive on the poly-l-lysine coated polymer for more than a week. The alignment of neurites on the astrocyte layer to the underlying topography decreases over time, and with groove width. Topographies with 12.5 or 25 microm lateral dimension appear optimal for the long-term alignment and can support myelination. We have shown for the first time that micro-topography can act through an overlaid astrocyte layer and results in aligned neurites in long-term culture and that these can be myelinated by endogenous oligodendrocytes.

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

    NASA Astrophysics Data System (ADS)

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

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

  6. Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides

    SciTech Connect

    Robakis, N.K.; Ramakrishna, N.; Wolfe, G.; Wisniewski, H.M.

    1987-06-01

    Deposits of amyloid fibers are found in large numbers in the walls of blood vessels and in neuritic plaques in the brains of patients with Alzheimer disease and adults with Down syndrome. The authors used the amino acid sequence of the amyloid peptide to synthesize oligonucleotide probes specific for the gene encoding this peptide. When a human brain cDNA library was screened with this probe, a clone was found with a 1.7-kilobase insert that contains a long open reading frame coding for 412 amino acid residues including the 28 amino acids of the amyloid peptide. RNA gel blots revealed that a 3.3-kilobase mRNA species was present in the brains of individuals with Alzheimer disease, with Down syndrome, or with not apparent neurological disorders. Southern blots showed that homologous genes are present in the genomic DNA of humans, rabbits, sheep, hamsters, and mice, suggesting that this gene has been conserved through mammalian evolution. Localization of the corresponding genomic sequences on human chromosome 21 suggest a genetic relationship between Alzheimer disease and Down syndrome, and it may explain the early appearance of large numbers of neuritic plaques in adult Down syndrome patients.

  7. A glial cell line-derived neurotrophic factor-secreting clone of the Schwann cell line SCTM41 enhances survival and fiber outgrowth from embryonic nigral neurons grafted to the striatum and to the lesioned substantia nigra.

    PubMed

    Wilby, M J; Sinclair, S R; Muir, E M; Zietlow, R; Adcock, K H; Horellou, P; Rogers, J H; Dunnett, S B; Fawcett, J W

    1999-03-15

    We have developed a novel Schwann cell line, SCTM41, derived from postnatal sciatic nerve cultures and have stably transfected a clone with a rat glial cell line-derived neurotrophic factor (GDNF) construct. Coculture with this GDNF-secreting clone enhances in vitro survival and fiber growth of embryonic dopaminergic neurons. In the rat unilateral 6-OHDA lesion model of Parkinson's disease, we have therefore made cografts of these cells with embryonic day 14 ventral mesencephalic grafts and assayed for effects on dopaminergic cell survival and process outgrowth. We show that cografts of GDNF-secreting Schwann cell lines improve the survival of intrastriatal embryonic dopaminergic neuronal grafts and improve neurite outgrowth into the host neuropil but have no additional effect on amphetamine-induced rotation. We next looked to see whether bridge grafts of GDNF-secreting SCTM41 cells would promote the growth of axons to their striatal targets from dopaminergic neurons implanted orthotopically into the 6-OHDA-lesioned substantia nigra. We show that such bridge grafts increase the survival of implanted embryonic dopaminergic neurons and promote the growth of axons through the grafts to the striatum.

  8. Semaphorin 7A promotes axon outgrowth through integrins and MAPKs.

    PubMed

    Pasterkamp, R Jeroen; Peschon, Jacques J; Spriggs, Melanie K; Kolodkin, Alex L

    2003-07-24

    Striking parallels exist between immune and nervous system cellular signalling mechanisms. Molecules originally shown to be critical for immune responses also serve neuronal functions, and similarly neural guidance cues can modulate immune function. We show here that semaphorin 7A (Sema7A), a membrane-anchored member of the semaphorin family of guidance proteins previously known for its immunomodulatory effects, can also mediate neuronal functions. Unlike many other semaphorins, which act as repulsive guidance cues, Sema7A enhances central and peripheral axon growth and is required for proper axon tract formation during embryonic development. Unexpectedly, Sema7A enhancement of axon outgrowth requires integrin receptors and activation of MAPK signalling pathways. These findings define a previously unknown biological function for semaphorins, identify an unexpected role for integrins and integrin-dependent intracellular signalling in mediating semaphorin responses, and provide a framework for understanding and interfering with Sema7A function in both immune and nervous systems.

  9. Tendon cell outgrowth rates and morphology associated with kevlar-49.

    PubMed

    Zimmerman, M; Gordon, K E

    1988-12-01

    A rat tendon cell model was used to evaluate the in vitro biocompatibility of kevlar-49. The cell response to kevlar was compared to carbon AS-4 and nylon sutures. Three trials were run and cell growth rates were statistically similar for all the materials tested. A separate experiment was conducted in which the same fiber materials were placed in the same Petri dish. Again, the rates were similar for each material. Finally, the cells were observed with a scanning electron microscope, and the three classic cell morphologies associated with this tendon cell model were observed. Also, cellular attachment to the fiber and cellular encapsulation of the fiber were identical for the three materials tested. Kevlar-49 proved to be comparable to carbon AS4 and nylon sutures in terms of cellular response and cell outgrowth rates.

  10. gamma-Aminobutyric acid (GABA): a fast excitatory transmitter which may regulate the development of hippocampal neurones in early postnatal life.

    PubMed

    Ben-Ari, Y; Tseeb, V; Raggozzino, D; Khazipov, R; Gaiarsa, J L

    1994-01-01

    The properties of neonatal GABAergic synapses were investigated in neurones of the hippocampal CA3 region. GABA, acting on GABAA receptors, provides most of the excitatory drive on immature CA3 pyramidal neurones at an early stage of development, whereas glutamatergic synapses (in particular, those mediated by AMPA receptors) are mostly quiescent. Thus, during the first postnatal week of life, bicuculline fully blocked spontaneous and evoked depolarising potentials, and GABAA receptor agonists depolarised CA3 pyramidal neurones. GABAA mediated currents also had a reduced sensitivity to benzodiazepines. In the presence of bicuculline, between P0 and P4, increasing the stimulus strength reveals an excitatory postsynaptic potential which is mostly mediated by NMDA receptors. During the same developmental period, pre- (but not post) synaptic GABAB inhibition is present. Intracellular injections of biocytin showed that the axonal network of the GABAergic interneurones is well developed at birth, whereas the pyramidal recurrent collaterals are only beginning to develop. Finally, chronic bicuculline treatment of hippocampal neurones in culture reduced the extent of neuritic arborisation, suggesting that GABA acts as a trophic factor in that period. In conclusion, it is suggested that during the first postnatal week of life, when excitatory inputs are still poorly developed, GABAA receptors provide the excitatory drive necessary for pyramidal cell outgrowth. Starting from the end of the first postnatal week of life, when excitatory inputs are well developed, GABA (acting on both GABAA and GABAB receptors) will hyperpolarise the CA3 pyramidal neurones and, as in the adult, will prevent excessive neuronal discharges. Our electrophysiological and morphological studies have shown that hippocampal GABAergic interneurones are in a unique position to modulate the development of CA3 pyramidal neurones. Developing neurones require a certain degree of membrane depolarisation, and a

  11. Proteomic analysis of differentiating neuroblastoma cells treated with sub-lethal neurite inhibitory concentrations of diazinon: Identification of novel biomarkers of effect

    SciTech Connect

    Harris, W.; Sachana, M.; Flaskos, J.; Hargreaves, A.J.

    2009-10-15

    In previous work we showed that sub-lethal levels of diazinon inhibited neurite outgrowth in differentiating N2a neuroblastoma cells. Western blotting analysis targeted at proteins involved in axon growth and stress responses, revealed that such exposure led to a reduction in the levels of neurofilament heavy chain, microtubule associated protein 1 B (MAP 1B) and HSP-70. The aim of this study was to apply the approach of 2 dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify novel biomarkers of effect. A number of proteins were found to be up-regulated compared to the control on silver-stained gels. These were classified in to 3 main groups of proteins: cytosolic factors, chaperones and the actin-binding protein cofilin, all of which are involved in cell differentiation, survival or metabolism. The changes observed for cofilin were further confirmed by quantitative Western blotting analysis with anti-actin and anti-cofilin antibodies. Indirect immunofluorescence staining with the same antibodies indicated that the microfilament network was disrupted in diazinon-treated cells. Our data suggest that microfilament organisation is disrupted by diazinon exposure, which may be related to increased cofilin expression.

  12. CB1 cannabinoid receptor-mediated neurite remodeling in mouse neuroblastoma N1E-115 cells.

    PubMed

    Zhou, D; Song, Z H

    2001-08-15

    The morphological remodeling of neuronal cells influences neurogenesis and brain functions. We hypothesize that psychoactive and neurotoxic effects of cannabinoids may be mediated, at least in part, by their morphoregulatory activities. In the present study, mouse neuroblastoma N1E-115 cells were used as an in vitro model to investigate cannabinoid-induced neurite remodeling effects and to identify the involvement of cannabinoid receptors in this neurite remodeling process. Using reverse transcription-polymerase chain reaction and immunofluorescence microscopy, the endogenously expressed CB1, but not CB2, cannabinoid receptors were detected in morphologically differentiated N1E-115 cells. Activation of these natively expressed CB1 cannabinoid receptors by cannabinoid agonist HU-210 led to a concentration-dependent inhibition of adenylate cyclase activity. Importantly, HU-210 treatment induced neurite retraction in a concentration-dependent manner. Pretreatment of N1E-115 cells with a CB1 antisense oligodeoxynucleotide (ODN) suppressed HU-210-induced inhibition of forskolin-stimulated cAMP accumulation, indicating that the knocking down of functional CB1 cannabinoid receptor expression was achieved. Antisense ODN pretreatment also abolished HU-210-induced neurite retraction, demonstrating the involvement of CB1 cannabinoid receptors in mediating the neurite remodeling effects of HU-210. In addition, reversing HU-210-induced intracellular cAMP declination by 8-Br-cAMP partially prevented HU-210-induced neurite retraction, indicating the involvement of cAMP-dependent signaling pathways in mediating the neurite remodeling function of CB1 cannabinoid receptors in N1E-115 cells. These data demonstrate that neurite remodeling is a newly discovered function of CB1 cannabinoid receptors. This morphoregulatory function of CB1 cannabinoid receptors might be a new mechanism that mediates the psychoactive and neurotoxic effects of cannabinoids in developing and adult brain.

  13. The serine/threonine kinase Ndr2 controls integrin trafficking and integrin-dependent neurite growth.

    PubMed

    Rehberg, Kati; Kliche, Stefanie; Madencioglu, Deniz A; Thiere, Marlen; Müller, Bettina; Meineke, Bernhard Manuel; Freund, Christian; Budinger, Eike; Stork, Oliver

    2014-04-09

    Integrins have been implicated in various processes of nervous system development, including proliferation, migration, and differentiation of neuronal cells. In this study, we show that the serine/threonine kinase Ndr2 controls integrin-dependent dendritic and axonal growth in mouse hippocampal neurons. We further demonstrate that Ndr2 is able to induce phosphorylation at the activity- and trafficking-relevant site Thr(788/789) of β1-integrin to stimulate the PKC- and CaMKII-dependent activation of β1-integrins, as well as their exocytosis. Accordingly, Ndr2 associates with integrin-positive early and recycling endosomes in primary hippocampal neurons and the surface expression of activated β1-integrins is reduced on dendrites of Ndr2-deficient neurons. The role of Ndr2 in dendritic differentiation is also evident in vivo, because Ndr2-null mutant mice show arbor-specific alterations of dendritic complexity in the hippocampus. This indicates a role of Ndr2 in the fine regulation of dendritic growth; in fact, treatment of primary neurons with Semaphorin 3A rescues Ndr2 knock-down-induced dendritic growth deficits but fails to enhance growth beyond control level. Correspondingly, Ndr2-null mutant mice show a Semaphorin 3A(-/-)-like phenotype of premature dendritic branching in the hippocampus. The results of this study show that Ndr2-mediated integrin trafficking and activation are crucial for neurite growth and guidance signals during neuronal development.

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

    PubMed

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

    2016-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  16. Neuritic regeneration and synaptic reconstruction induced by withanolide A

    PubMed Central

    Kuboyama, Tomoharu; Tohda, Chihiro; Komatsu, Katsuko

    2005-01-01

    We investigated whether withanolide A (WL-A), isolated from the Indian herbal drug Ashwagandha (root of Withania somnifera), could regenerate neurites and reconstruct synapses in severely damaged neurons. We also investigated the effect of WL-A on memory-deficient mice showing neuronal atrophy and synaptic loss in the brain. Axons, dendrites, presynapses, and postsynapses were visualized by immunostaining for phosphorylated neurofilament-H (NF-H), microtubule-associated protein 2 (MAP2), synaptophysin, and postsynaptic density-95 (PSD-95), respectively. Treatment with Aβ(25–35) (10 μM) induced axonal and dendritic atrophy, and pre- and postsynaptic loss in cultured rat cortical neurons. Subsequent treatment with WL-A (1 μM) induced significant regeneration of both axons and dendrites, in addition to the reconstruction of pre- and postsynapses in the neurons. WL-A (10 μmol kg−1 day−1, for 13 days, p.o.) recovered Aβ(25–35)-induced memory deficit in mice. At that time, the decline of axons, dendrites, and synapses in the cerebral cortex and hippocampus was almost recovered. WL-A is therefore an important candidate for the therapeutic treatment of neurodegenerative diseases, as it is able to reconstruct neuronal networks. PMID:15711595

  17. Embryoid Body-Explant Outgrowth Cultivation from Induced Pluripotent Stem Cells in an Automated Closed Platform

    PubMed Central

    Tone, Hiroshi; Yoshioka, Saeko; Akiyama, Hirokazu; Nishimura, Akira; Ichimura, Masaki; Nakatani, Masaru; Kiyono, Tohru

    2016-01-01

    Automation of cell culture would facilitate stable cell expansion with consistent quality. In the present study, feasibility of an automated closed-cell culture system “P 4C S” for an embryoid body- (EB-) explant outgrowth culture was investigated as a model case for explant culture. After placing the induced pluripotent stem cell- (iPSC-) derived EBs into the system, the EBs successfully adhered to the culture surface and the cell outgrowth was clearly observed surrounding the adherent EBs. After confirming the outgrowth, we carried out subculture manipulation, in which the detached cells were simply dispersed by shaking the culture flask, leading to uniform cell distribution. This enabled continuous stable cell expansion, resulting in a cell yield of 3.1 × 107. There was no evidence of bacterial contamination throughout the cell culture experiments. We herewith developed the automated cultivation platform for EB-explant outgrowth cells. PMID:27648449

  18. Acceleration of axonal outgrowth in rat sciatic nerve at one week after axotomy.

    PubMed

    Jacob, J M; McQuarrie, I G

    1993-03-01

    Following injury of sciatic motor axons in the rat, the rate of axonal outgrowth is faster if there has been a prior "conditioning" axotomy. The acceleration of outgrowth is due to an acceleration of SCb, the rate [slow (SC)] component of axonal transport that carries cytomatrix proteins; this occurs throughout the axon by 7 days after the conditioning axotomy (Jacob and McQuarrie, 1991a, J. Neurobiol. 22:570-583). To further characterize the conditioning lesion effect (CLE), it is important to know (1) the minimum effective conditioning interval (time between conditioning and testing lesions), (2) whether the cell body reaction is required, and (3) whether outgrowth accelerates after a single axotomy. Outgrowth distances were measured by radiolabeling all newly synthesized neuronal proteins and detecting those carried to growth cones by fast axonal transport. When the conditioning and testing lesions were made simultaneously (0 day conditioning interval), there was no CLE. With a conditioning interval of 3 days, there was a shortening of the initial delay (before the onset of outgrowth) without a change in outgrowth rate. With conditioning intervals of 7, 14, and 21 days, the rates of outgrowth were increased by 8%, 22%, and 11%, respectively. To determine whether the cell body reaction to axotomy is necessary for the CLE, a nonaxotomizing stimulus to axonal growth (partial denervation) was used in place of a conditioning axotomy. This had no effect on the rate of outgrowth from a testing lesion made 14 days later. Finally, we examined the possibility that outgrowth accelerates after a single lesion. Outgrowth was faster at 6-9 days after axotomy than at 3-6 days (p < 0.001), and accelerated further at 9-12 days (p < 0.001). We conclude that (1) the shortest effective conditioning interval is 3 days; (2) the cell body reaction is necessary for the CLE; (3) axonal outgrowth from a single axotomy accelerates in concert with the anabolic phase of the cell body

  19. Calsyntenin-3 C-terminal fragment accumulates in dystrophic neurites surrounding aβ plaques in tg2576 mouse and Alzheimer disease brains: its neurotoxic role in mediating dystrophic neurite formation.

    PubMed

    Uchida, Yoko; Gomi, Fujiya; Murayama, Shigeo; Takahashi, Hiroshi

    2013-05-01

    Dystrophic neurites surrounding β-amyloid (Aβ) plaques precede neuronal death in Alzheimer disease. These neuritic alterations may be one of the initial stages for synaptic loss and dysfunction. However, intracellular pathways that cause local disruption of neuronal processes by Aβ remain to be fully elucidated. The identification of Aβ-induced genes that mediate neuritic pathology would provide considerable insight into the mechanisms of Alzheimer's disease. Previously, we reported that selective up-regulation of calsyntenin-3 (Cst-3) by Aβ and accumulation of neurotoxic Cst-3 in dystrophic neurites surrounding Aβ plaques may lead to local disruption of these neurites. Like amyloid precursor protein, Cst-3 undergoes two-step proteolytic processing: the primary cleavage with α-secretase generates an N-terminal ectodomain and a C-terminal fragment (CTF). The CTF is subsequently cleaved into p3 peptide and an intracellular domain via γ-secretase. It would be interesting to know whether accumulated Cst-3 in dystrophic neurites surrounding Aβ plaques is the full-length version or a CTF. Herein, we show that the CTF but not full-length Cst-3 accumulated in dystrophic neurites surrounding Aβ plaques in Tg2576 mouse and Alzheimer disease brains. In vitro experiments with Cst-3 fragments have revealed that only the CTF resulted in acceleration of neuronal death. These results indicate that accumulation of the neurotoxic CTF in neurites surrounding Aβ plaques may lead to local disruption of neuronal processes and development of dystrophic neurites.

  20. Insensitivity of ciliary outgrowth in situ by Stentor to far UV.

    PubMed

    Burchill, B R

    1979-10-01

    Outgrowth of membranellar cilia in situ by the ciliate protozoan Stentor coeruleus is not affected by either far UV or by postirradiation exposure to cycloheximide, while UV does affect the outgrowth of membranellar cilia during the more elaborate process of oral regeneration in this ciliate, and cycloheximide prolongs the UV effect. These results suggest that direct damage to outgrowing ciliary shafts cannot explain the UV sensitivity of oral regeneration.

  1. Early exposure to ethanol but not red wine at the same alcohol concentration induces behavioral and brain neurotrophin alterations in young and adult mice.

    PubMed

    Fiore, Marco; Laviola, Giovanni; Aloe, Luigi; di Fausto, Veronica; Mancinelli, Rosanna; Ceccanti, Mauro

    2009-01-01

    Ethanol exposure during pregnancy is one of the major causes of mental retardation in western countries by inducing fetal-alcohol-like-syndromes. Red wine is known to contain ethanol but also compounds with putative antioxidant properties. It has also been shown that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are severely affected by ethanol during prenatal and postnatal life. The aim of the current study was to investigate in male CD1 mice brain alterations in NGF and BDNF due to chronic early exposure to ethanol solution (11 vol%) or to red wine at the same alcohol concentration starting from 60 days before pregnancy up to pups weaning. Data revealed no differences between groups of dams in pregnancy duration, neither in pups delivery, pups mortality and sex ratio. Data also showed that adult animals exposed to only ethanol had disrupted levels of both NGF and BDNF in the hippocampus and other brain areas. This profile was associated with impaired ChAT immunopositivity in the septum and Nuclei Basalis and with altered cognition and emotional behavior. Quite interestingly mice exposed to red wine had no change in the behavior or in ChAT immunopositivity but a decrease in hippocampal BDNF and a mild NGF decrease in the cortex. Also NGF-induced neuritic outgrowth in PC-12 cells was still present when exposed to red wine but not when exposed to ethanol solution only. Data suggest differences in ethanol-induced neurotoxicity between red wine and ethanol solution only.

  2. Gene dosage-dependent rescue of HSP neurite defects in SPG4 patients’ neurons

    PubMed Central

    Havlicek, Steven; Kohl, Zacharias; Mishra, Himanshu K.; Prots, Iryna; Eberhardt, Esther; Denguir, Naime; Wend, Holger; Plötz, Sonja; Boyer, Leah; Marchetto, Maria C.N.; Aigner, Stefan; Sticht, Heinrich; Groemer, Teja W.; Hehr, Ute; Lampert, Angelika; Schlötzer-Schrehardt, Ursula; Winkler, Jürgen; Gage, Fred H.; Winner, Beate

    2014-01-01

    The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients. PMID:24381312

  3. Gene dosage-dependent rescue of HSP neurite defects in SPG4 patients' neurons.

    PubMed

    Havlicek, Steven; Kohl, Zacharias; Mishra, Himanshu K; Prots, Iryna; Eberhardt, Esther; Denguir, Naime; Wend, Holger; Plötz, Sonja; Boyer, Leah; Marchetto, Maria C N; Aigner, Stefan; Sticht, Heinrich; Groemer, Teja W; Hehr, Ute; Lampert, Angelika; Schlötzer-Schrehardt, Ursula; Winkler, Jürgen; Gage, Fred H; Winner, Beate

    2014-05-15

    The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients.

  4. Micropatterned coumarin polyester thin films direct neurite orientation.

    PubMed

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

    2014-11-26

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

  5. Bingham-NODDI: Mapping anisotropic orientation dispersion of neurites using diffusion MRI.

    PubMed

    Tariq, Maira; Schneider, Torben; Alexander, Daniel C; Gandini Wheeler-Kingshott, Claudia A; Zhang, Hui

    2016-06-01

    This paper presents Bingham-NODDI, a clinically-feasible technique for estimating the anisotropic orientation dispersion of neurites. Direct quantification of neurite morphology on clinical scanners was recently realised by a diffusion MRI technique known as neurite orientation dispersion and density imaging (NODDI). However in its current form NODDI cannot estimate anisotropic orientation dispersion, which is widespread in the brain due to common fanning and bending of neurites. This work proposes Bingham-NODDI that extends the NODDI formalism to address this limitation. Bingham-NODDI characterises anisotropic orientation dispersion by utilising the Bingham distribution to model neurite orientation distribution. The new model estimates the extent of dispersion about the dominant orientation, separately along the primary and secondary dispersion orientations. These estimates are subsequently used to estimate the overall dispersion about the dominant orientation and the dispersion anisotropy. We systematically evaluate the ability of the new model to recover these key parameters of anisotropic orientation dispersion with standard NODDI protocol, both in silico and in vivo. The results demonstrate that the parameters of the proposed model can be estimated without additional acquisition requirements over the standard NODDI protocol. Thus anisotropic dispersion can be determined and has the potential to be used as a marker for normal brain development and ageing or in pathology. We additionally find that the original NODDI model is robust to the effects of anisotropic orientation dispersion, when the quantification of anisotropic dispersion is not of interest.

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

    PubMed Central

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

    2007-01-01

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

  7. Pharmacological inhibition of caspase-8 limits lung tumour outgrowth

    PubMed Central

    Terlizzi, Michela; Di Crescenzo, Vincenzo Giuseppe; Perillo, Giuseppe; Galderisi, Antonio; Pinto, Aldo; Sorrentino, Rosalinda

    2015-01-01

    Background and Purpose Lung cancer is one of the leading causes of cancer death worldwide. Despite advances in therapy, conventional therapy is still the main treatment and has a high risk of chemotherapy resistance. Caspase-8 is involved in cell death and is a recognized marker for poor patient prognosis. Experimental Approach To elucidate the role of caspase-8 in lung carcinoma, we used human samples of non-small cell lung cancer (NSCLC) and a mouse model of carcinogen-induced lung cancer. Key Results Healthy and cancerous NSCLC samples had similar levels of the active form of caspase-8. Similarly, lung tumour-bearing mice had high levels of the active form of caspase-8. Pharmacological inhibition of caspase-8 by z-IETD-FMK robustly reduced tumour outgrowth and this was closely associated with a reduction in the release of pro-inflammatory cytokines, IL-6, TNF-α, IL-18, IL-1α, IL-33, but not IL-1β. Furthermore, inhibition of caspase-8 reduced the recruitment of innate suppressive cells, such as myeloid-derived suppressor cells, but not of regulatory T cells to lungs of tumour-bearing mice. However, despite the well-known role of caspase-8 in cell death, the apoptotic cascade (caspase-3, caspase-9 and Bcl-2 dependent) was not active in lungs of z-IETD-treated tumour-bearing mice, but instead higher levels of the short segment of c-FLIP (c-FLIPs) were detected. Similarly, human healthy lung samples had higher levels of c-FLIPs than cancerous samples. Conclusions and Implications Our data suggest that caspase-8 is an important orchestrator of cancer-associated inflammation and the presence of short segment of c-FLIP determines whether caspase-8 induces tumour proliferation or tumour arrest/regression in the lung. PMID:25917370

  8. Mesenchymal Stromal Cells Promote Axonal Outgrowth Alone and Synergistically with Astrocytes via tPA

    PubMed Central

    Qian, Jian-Yong; Chopp, Michael

    2016-01-01

    We reported that mesenchymal stromal cells (MSCs) enhance neurological recovery from experimental stroke and increase tissue plasminogen activator (tPA) expression in astrocytes. Here, we investigate mechanisms by which tPA mediates MSC enhanced axonal outgrowth. Primary murine neurons and astrocytes were isolated from wild-type (WT) and tPA-knockout (KO) cortices of embryos. Mouse MSCs (WT) were purchased from Cognate Inc. Neurons (WT or KO) were seeded in soma side of Xona microfluidic chambers, and astrocytes (WT or KO) and/or MSCs in axon side. The chambers were cultured as usual (normoxia) or subjected to oxygen deprivation. Primary neurons (seeded in plates) were co-cultured with astrocytes and/or MSCs (in inserts) for Western blot. In chambers, WT axons grew significantly longer than KO axons and exogenous tPA enhanced axonal outgrowth. MSCs increased WT axonal outgrowth alone and synergistically with WT astrocytes at both normoxia and oxygen deprivation conditions. The synergistic effect was inhibited by U0126, an ERK inhibitor, and receptor associated protein (RAP), a low density lipoprotein receptor related protein 1 (LRP1) ligand antagonist. However, MSCs exerted neither individual nor synergistic effects on KO axonal outgrowth. Western blot showed that MSCs promoted astrocytic tPA expression and increased neuronal tPA alone and synergistically with astrocytes. Also, MSCs activated neuronal ERK alone and synergistically with astrocytes, which was inhibited by RAP. We conclude: (1) MSCs promote axonal outgrowth via neuronal tPA and synergistically with astrocytic tPA; (2) neuronal tPA is critical to observe the synergistic effect of MSC and astrocytes on axonal outgrowth; and (3) tPA mediates MSC treatment-induced axonal outgrowth through the LRP1 receptor and ERK. PMID:27959956

  9. Mechanism of Inhibition of Bacillus anthracis Spore Outgrowth by the Lantibiotic Nisin

    PubMed Central

    2011-01-01

    The lantibiotic nisin inhibits growth of vegetative Gram-positive bacteria by binding to lipid II, which disrupts cell wall biosynthesis and facilitates pore formation. Nisin also inhibits the outgrowth of bacterial spores, including spores of Bacillus anthracis, whose structural and biochemical properties are fundamentally different from those of vegetative bacteria. The molecular basis of nisin inhibition of spore outgrowth had not been identified, as previous studies suggested that inhibition of spore outgrowth involved either covalent binding to a spore target or loss of membrane integrity; disruption of cell wall biosynthesis via binding to lipid II had not been investigated. To provide insights into the latter possibility, the effects of nisin were compared with those of vancomycin, another lipid II binding antibiotic that inhibits cell wall biosynthesis but does not form pores. Nisin and vancomycin both inhibited the replication of vegetative cells, but only nisin inhibited the transition from a germinated spore to a vegetative cell. Moreover, vancomycin prevented nisin’s activity in competition studies, suggesting that the nisin-lipid II interaction is important for inhibition of spore outgrowth. In experiments with fluorescently labeled nisin, no evidence was found for a covalent mechanism for inhibition of spore outgrowth. Interestingly, mutants in the hinge region (N20P/M21P and M21P/K22P) that still bind lipid II but cannot form pores had potent antimicrobial activity against vegetative B. anthracis cells but did not inhibit spore outgrowth. Therefore, pore formation is essential for the latter activity but not the former. Collectively, these studies suggest that nisin utilizes lipid II as the germinated spore target during outgrowth inhibition and that nisin-mediated membrane disruption is essential to inhibit spore development into vegetative cells. PMID:21517116

  10. Transient neurites of retinal horizontal cells exhibit columnar tiling via homotypic interactions.

    PubMed

    Huckfeldt, Rachel M; Schubert, Timm; Morgan, Josh L; Godinho, Leanne; Di Cristo, Graziella; Huang, Z Josh; Wong, Rachel O L

    2009-01-01

    Sensory neurons with common functions are often nonrandomly arranged and form dendritic territories that show little overlap, or tiling. Repulsive homotypic interactions underlie such patterns in cell organization in invertebrate neurons. It is unclear how dendro-dendritic repulsive interactions can produce a nonrandom distribution of cells and their spatial territories in mammalian retinal horizontal cells, as mature horizontal cell dendrites overlap substantially. By imaging developing mouse horizontal cells, we found that these cells transiently elaborate vertical neurites that form nonoverlapping columnar territories on reaching their final laminar positions. Targeted cell ablation revealed that the vertical neurites engage in homotypic interactions that result in tiling of neighboring cells before the establishment of their dendritic fields. This developmental tiling of transient neurites correlates with the emergence of a nonrandom distribution of the cells and could represent a mechanism that organizes neighbor relationships and territories of neurons before circuit assembly.

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

    SciTech Connect

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

    2008-02-12

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

  12. Preparation of primary neurons for visualizing neurites in a frozen-hydrated state using cryo-electron tomography.

    PubMed

    Shahmoradian, Sarah H; Galiano, Mauricio R; Wu, Chengbiao; Chen, Shurui; Rasband, Matthew N; Mobley, William C; Chiu, Wah

    2014-02-12

    Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts. Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.

  13. Neuritic Plaques and Cerebrovascular Amyloid in Alzheimer Disease are Antigenically Related

    NASA Astrophysics Data System (ADS)

    Wong, Caine W.; Quaranta, Vito; Glenner, George G.

    1985-12-01

    A synthetic peptide (Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr), homologous to the amino terminus of a protein purified from cerebrovascular amyloid (β protein), induced antibodies in BALB/c mice that were used immunohistochemically to stain not only amyloid-laden cerebral vessels but neuritic plaques as well. These findings suggest that the amyloid in neuritic plaques shares antigenic determinants with β protein of cerebral vessels. Since the amino acid compositions of plaque amyloid and cerebrovascular amyloid are similar, it is likely that plaque amyloid also consists of β protein. This possibility suggests a model for the pathogenesis of Alzheimer disease involving β protein.

  14. Fish 'tails' result from outgrowth and reduction of two separate ancestral tails.

    PubMed

    Sallan, Lauren

    2016-12-05

    The symmetrical, flexible teleost fish 'tail' has been a prime example of recapitulation - evolutionary change (phylogeny) mirrored in development (ontogeny). Paleozoic ray-finned fishes (Actinopterygii), relatives of teleosts, exhibited ancestral scale-covered tails curved over their caudal fins. For over 150 years, this arrangement was thought to be retained in teleost larva and overgrown, mirroring an ancestral transformation series. New ontogenetic data for the 350-million-year-old teleost relative Aetheretmon overturns this long-held hypothesis. The ancestral state consists of two outgrowths with distinct organizers and growth trajectories; a lower median fin turned caudal fin, and an upper vertebrae-bearing tail, equivalent to that of tetrapods. These two tails appear at a shared developmental stage in Aetheretmon, teleosts and all living actinopterygians. Ontogeny does not recapitulate phylogeny; instead, differential outgrowth determines final morphology. In Aetheretmon and other Paleozoic fishes, the vertebrae-bearing tail continues to grow beyond the caudal fin. In teleosts, and some others, a stunted tail is eclipsed by the upward-expanding caudal fin, rendering a once ventral body margin as the terminus. The double tail likely reflects the ancestral state for bony fishes. Many tetrapods and non-teleost actinopterygians have undergone body elongation through tail outgrowth extension, by mechanisms likely shared with distal limbs. Teleosts have gone to the other extreme; losing tail outgrowth for functional reasons. Recognition of the tail as a limb-like outgrowth has important implications for the evolution of vertebrate form.

  15. Serum and CNTF stimulate oligodendroglia and reduce fiber outgrowth from striatal cultures.

    PubMed

    Dahl-Jørgensen, A; Ostergaard, K; Pedersen, E B; Zimmer, J

    1999-05-01

    Organotypic slice cultures of newborn rat striatal tissue displayed an exceptionally dense and fasciculated outgrowth of GABAergic fibers when grown in a chemically defined medium, compared to serum-containing medium. The enhanced fiber growth was not the result of an increased density of GABAergic neurons in the cultures, but coincided with a marked reduction of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-immunoreactive cells within and around the cultures. An inverse, causal relationship between the number of CNPase-positive cells, presumably of oligodendroglial lineage, and GABAergic fiber outgrowth was further evidenced by the observation that addition of ciliary neurotrophic factor (CNTF) to the chemically defined medium resulted in both an increase in CNPase-positive cells and a decrease in GABAergic fiber outgrowth. The observations suggest that CNTF and serum indirectly inhibit axonal growth by stimulating oligodendroglial cells.

  16. Cytokinins Are Initial Targets of Light in the Control of Bud Outgrowth1[OPEN

    PubMed Central

    Girault, Tiffanie; Barbier, François; Péron, Thomas; Pěnčík, Aleš; Sakr, Soulaiman; Lothier, Jérémy

    2016-01-01

    Bud outgrowth is controlled by environmental and endogenous factors. Through the use of the photosynthesis inhibitor norflurazon and of masking experiments, evidence is given here that light acts mainly as a morphogenic signal in the triggering of bud outgrowth and that initial steps in the light signaling pathway involve cytokinins (CKs). Indeed, in rose (Rosa hybrida), inhibition of bud outgrowth by darkness is suppressed solely by the application of CKs. In contrast, application of sugars has a limited effect. Exposure of plants to white light (WL) induces a rapid (after 3–6 h of WL exposure) up-regulation of CK synthesis (RhIPT3 and RhIPT5), of CK activation (RhLOG8), and of CK putative transporter RhPUP5 genes and to the repression of the CK degradation RhCKX1 gene in the node. This leads to the accumulation of CKs in the node within 6 h and in the bud at 24 h and to the triggering of bud outgrowth. Molecular analysis of genes involved in major mechanisms of bud outgrowth (strigolactone signaling [RwMAX2], metabolism and transport of auxin [RhPIN1, RhYUC1, and RhTAR1], regulation of sugar sink strength [RhVI, RhSUSY, RhSUC2, and RhSWEET10], and cell division and expansion [RhEXP and RhPCNA]) reveal that, when supplied in darkness, CKs up-regulate their expression as rapidly and as intensely as WL. Additionally, up-regulation of CKs by WL promotes xylem flux toward the bud, as evidenced by Methylene Blue accumulation in the bud after CK treatment in the dark. Altogether, these results suggest that CKs are initial components of the light signaling pathway that controls the initiation of bud outgrowth. PMID:27462085

  17. Reduced cell cohesiveness of outgrowths from eccrine sweat glands delays wound closure in elderly skin.

    PubMed

    Rittié, Laure; Farr, Elyssa A; Orringer, Jeffrey S; Voorhees, John J; Fisher, Gary J

    2016-10-01

    Human skin heals more slowly in aged vs. young adults, but the mechanism for this delay is unclear. In humans, eccrine sweat glands (ESGs) and hair follicles underlying wounds generate cohesive keratinocyte outgrowths that expand to form the new epidermis. Here, we compared the re-epithelialization of partial-thickness wounds created on the forearm of healthy young (< 40 yo) and aged (> 70 yo) adults. Our results confirm that the outgrowth of cells from ESGs is a major feature of repair in young skin. Strikingly, in aged skin, although ESG density is unaltered, less than 50% of the ESGs generate epithelial outgrowths during repair (vs. 100% in young). Surprisingly, aging does not alter the wound-induced proliferation response in hair follicles or ESGs. Instead, there is an overall reduced cohesiveness of keratinocytes in aged skin. Reduced cell-cell cohesiveness was most obvious in ESG-derived outgrowths that, when present, were surrounded by unconnected cells in the scab overlaying aged wounds. Reduced cell-cell contact persisted during the repair process, with increased intercellular spacing and reduced number of desmosomes. Together, reduced outgrowths of ESG (i) reduce the initial number of cells participating in epidermal repair, (ii) delay wound closure, and (iii) lead to a thinner repaired epidermis in aged vs. young skin. Failure to form cohesive ESG outgrowths may reflect impaired interactions of keratinocytes with the damaged ECM in aged skin. Our findings provide a framework to better understand the mediators of delayed re-epithelialization in aging and further support the importance of ESGs for the repair of human wounds.

  18. Effects of antidepressant drugs on synaptic protein levels and dendritic outgrowth in hippocampal neuronal cultures.

    PubMed

    Seo, Mi Kyoung; Lee, Chan Hong; Cho, Hye Yeon; Lee, Jung Goo; Lee, Bong Ju; Kim, Ji Eun; Seol, Wongi; Kim, Young Hoon; Park, Sung Woo

    2014-04-01

    The alteration of hippocampal plasticity has been proposed to play a critical role in both the pathophysiology and treatment of depression. In this study, the ability of different classes of antidepressant drugs (escitalopram, fluoxetine, paroxetine, sertraline, imipramine, tranylcypromine, and tianeptine) to mediate the expression of synaptic proteins and dendritic outgrowth in rat hippocampal neurons was investigated under toxic conditions induced by B27 deprivation, which causes hippocampal cell death. Postsynaptic density protein-95 (PSD-95), brain-derived neurotrophic factor (BDNF), and synaptophysin (SYP) levels were evaluated using Western blot analyses. Additionally, dendritic outgrowth was examined to determine whether antidepressant drugs affect the dendritic morphology of hippocampal neurons in B27-deprived cultures. Escitalopram, fluoxetine, paroxetine, sertraline, imipramine, tranylcypromine, and tianeptine significantly prevented B27 deprivation-induced decreases in levels of PSD-95, BDNF, and SYP. Moreover, the independent application of fluoxetine, paroxetine, and sertraline significantly increased levels of BDNF under normal conditions. All antidepressant drugs significantly increased the total outgrowth of hippocampal dendrites under B27 deprivation. Specific inhibitors of calcium/calmodulin kinase II (CaMKII), KN-93, protein kinase A (PKA), H-89, or phosphatidylinositol 3-kinase (PI3K), LY294002, significantly decreased the effects of antidepressant drugs on dendritic outgrowth, whereas this effect was observed only with tianeptine for the PI3K inhibitor. Taken together, these results suggest that certain antidepressant drugs can enhance synaptic protein levels and encourage dendritic outgrowth in hippocampal neurons. Furthermore, effects on dendritic outgrowth likely require CaMKII, PKA, or PI3K signaling pathways. The observed effects may be may be due to chronic treatment with antidepressant drugs.

  19. Notch activation induces neurite remodeling and functional modifications in SH-SY5Y neuronal cells.

    PubMed

    Ferrari-Toninelli, Giulia; Bonini, Sara Anna; Uberti, Daniela; Napolitano, Francesco; Stante, Maria; Santoro, Federica; Minopoli, Giuseppina; Zambrano, Nicola; Russo, Tommaso; Memo, Maurizio

    2009-05-01

    Notch proteins are definitely recognized as key regulators of the neuronal fate during embryo development, but their function in the adult brain is still largely unknown. We have previously demonstrated that Notch pathway stimulation increases microtubules stability followed by the remodeling of neuronal morphology with neurite varicosities loss, thicker neuritis, and enlarged growth cones. Here we show that the neurite remodeling is a dynamic event, dependent on transcription and translation, and with functional implications. Exposure of differentiated human SH-SY5Y neuroblastoma cells to the Notch ligand Jagged1 induces varicosities loss all along the neurites, accompanied by the redistribution of presynaptic vesicles and the decrease in neurotransmitters release. As evaluated by time lapse digital imaging, dynamic changes in neurite morphology were rapidly reversible and dependent on the activation of the Notch signaling pathway. In fact, it was prevented by the inhibition of the proteolytic gamma-secretase enzyme or the transcription machinery, and was mimicked by the transfection of the intracellular domain of Notch. One hour after treatment with Jagged1, several genes were downregulated. Many of these genes encode proteins that are known to be involved in protein synthesis. These data suggest that in adult neurons, Notch pathway activates a transcriptional program that regulates the equilibrium between varicosities formation and varicosities loss in the neuronal presynaptic compartment involving the expression and redistribution of both structural and functional proteins.

  20. White matter microstructure pathology in classic galactosemia revealed by neurite orientation dispersion and density imaging.

    PubMed

    Timmers, Inge; Zhang, Hui; Bastiani, Matteo; Jansma, Bernadette M; Roebroeck, Alard; Rubio-Gozalbo, M Estela

    2015-03-01

    White matter abnormalities have been observed in patients with classic galactosemia, an inborn error of galactose metabolism. However, magnetic resonance imaging (MRI) data collected in the past were generally qualitative in nature. Our objective was to investigate white matter microstructure pathology and examine correlations with outcome and behaviour in this disease, by using multi-shell diffusion weighted imaging. In addition to standard diffusion tensor imaging (DTI), neurite orientation dispersion and density imaging (NODDI) was used to estimate density and orientation dispersion of neurites in a group of eight patients (aged 16-21 years) and eight healthy controls (aged 15-20 years). Extensive white matter abnormalities were found: neurite density index (NDI) was lower in the patient group in bilateral anterior areas, and orientation dispersion index (ODI) was increased mainly in the left hemisphere. These specific regional profiles are in agreement with the cognitive profile observed in galactosemia, showing higher order cognitive impairments, and language and motor impairments, respectively. Less favourable white matter properties correlated positively with age and age at onset of diet, and negatively with behavioural outcome (e.g. visual working memory). To conclude, this study provides evidence of white matter pathology regarding density and dispersion of neurites in these patients. The results are discussed in light of suggested pathophysiological mechanisms.

  1. Model of outgrowths in the spiral galaxies NGC 4921 and NGC 7049 and the origin of spiral arms

    NASA Astrophysics Data System (ADS)

    Carlqvist, Per

    2013-02-01

    NGC 4921 and 7049 are two spiral galaxies presenting narrow, distinct dust features. A detailed study of the morphology of those features has been carried out using Hubble Space Telescope archival images. NGC 4921 shows a few but well-defined dust arms midway to its centre while NGC 7049 displays many more dusty features, mainly collected within a ring-shaped formation. Numerous dark and filamentary structures, called outgrowths, are found to protrude from the dusty arms in both galaxies. The outgrowths point both outwards and inwards in the galaxies. Mostly they are found to be V-shaped or Y-shaped with the branches connected to dark arm filaments. Often the stem of the Y appears to consist of intertwined filaments. Remarkably, the outgrowths show considerable similarities to elephant trunks in H ii regions. A model of the outgrowths, based on magnetized filaments, is proposed. The model provides explanations of both the shapes and orientations of the outgrowths. Most important, it can also give an account for their intertwined structures. It is found that the longest outgrowths are confusingly similar to dusty spiral arms. This suggests that some of the outgrowths can develop into such arms. The time-scale of the development is estimated to be on the order of the rotation period of the arms or shorter. Similar processes may also take place in other spiral galaxies. If so, the model of the outgrowths can offer a new approach to the old winding problem of spiral arms.

  2. The p75 neurotrophin receptor promotes Aβ-induced neuritic dystrophy in vitro and in vivo

    PubMed Central

    Knowles, Juliet; Rajadas, Jayakumar; Nguyen, Thuy-Vi V.; Yang, Tao; LeMieux, Melburne C.; Griend, Lilith Vander; Ishikawa, Chihiro; Massa, Stephen M.; Wyss-Coray, Tony; Longo, Frank M.

    2009-01-01

    Oligomeric forms of amyloid-β(1–42) (Aβ) are thought to play a causal role in Alzheimer’s disease (AD) and the p75 neurotrophin receptor (p75NTR) has been implicated in Aβ-induced neurodegeneration. To further define the functions of p75NTR in AD, we examined the interaction of oligomeric Aβ with p75NTR, and the effects of that interaction on neurite integrity in neuron cultures and in a chronic AD mouse model. Atomic force microscopy was used to ascertain the aggregated state of Aβ, and fluorescence resonance energy transfer (FRET) analysis revealed that Aβ oligomers interact with the extracellular domain of p75NTR. In vitro studies of Aβ-induced death in neuron cultures isolated from wildtype and p75NTR −/− mice, in which the p75NTR extracellular domain is deleted, showed reduced sensitivity of mutant cells to Aβ-induced cell death. Interestingly, Aβ-induced neuritic dystrophy and activation of c-Jun, a known mediator of Aβ-induced deleterious signaling, were completely prevented in p75NTR −/− neuron cultures. Thy1-hAPPLond/Swe X p75NTR−/− mice exhibited significantly diminished hippocampal neuritic dystrophy and complete reversal of basal forebrain cholinergic neurite degeneration relative to those expressing wild type p75NTR. Aβ levels were not affected, suggesting that removal of p75NTR extracellular domain reduced the ability of excess Aβ to promote neuritic degeneration. These findings indicate that while p75NTR likely does not mediate all Aβ effects, it does play a significant role in enabling Aβ-induced neurodegeneration in vitro and in vivo, establishing p75NTR as an important therapeutic target for AD. PMID:19710315

  3. Lack of variant specific CD8+ T-cell response against mutant and pre-existing variants leads to outgrowth of particular clones in acute hepatitis C

    PubMed Central

    2013-01-01

    Background CTL escape mutations have been described during acute hepatitis C in patients who developed chronic disease later on. Our aim was to investigate the mutual relationship between HCV specific CD8+ T cells and evolution of the viral sequence during early acute HCV infection. Results We sequenced multiple clones of NS3 1406 epitope in 4 HLA-A*02 patients with acute hepatitis C genotype 1b infection. Pentamers specific for the variants were used to monitor the corresponding CD8+ T cell response. We observed outgrowth of mutations, which induced only a weak and thus potentially insufficient CD8+ T cell response. In one patient we observed outgrowth of variant epitopes with similarities to a different genotype rather than de novo mutations most probably due to a lack of responsiveness to these likely pre-existing variants. We could show that in acute hepatitis C CTL escape mutations occur much earlier than demonstrated in previous studies. Conclusions The adaption of the virus to a new host is characterized by a high and rapid variability in epitopes under CD8+ T cell immune pressure. This adaption takes place during the very early phase of acute infection and strikingly some sequences were reduced below the limit of detection at some time points but were detected at high frequency again at later time points. Independent of the observed variability, HCV-specific CD8+ T cell responses decline and no adaption to different or new antigens during the course of infection could be detected. PMID:24073713

  4. A growth-promoting influence from the mesonephros during limb outgrowth.

    PubMed

    Geduspan, J S; Solursh, M

    1992-05-01

    It has been suggested that the mesonephros has a role in normal limb development. This hypothesis was directly tested by removing the mesonephros adjacent to the presumptive limb region of stage 12-18 chick embryos using microsurgery or laser ablation. The experimental manipulation resulted in reduced limb outgrowth on the operated side. The poor limb outgrowth was correlated with either the lack of or the presence of a rudimentary mesonephros on the operated side. Furthermore, the presence of nephric tissue in limb bud organ culture enhanced growth and morphological differentiation of cartilage formed in culture. In vivo, the influence of the mesonephros resulted in significantly higher cell proliferation in the adjoining medial half of the limb mesoderm compared with the lateral half. The removal of the mesonephros adjoining the prospective limb region reduced the number of dividing cells in the medial mesoderm. The higher proliferation in the medial limb mesoderm is significant to limb outgrowth since grafting exp