PAK5, a New Brain-Specific Kinase, Promotes Neurite Outgrowth in N1E-115 Cells

PubMed Central

Dan, Chuntao; Nath, Niharika; Liberto, Muriel; Minden, Audrey

2002-01-01

We have characterized a new member of the mammalian PAK family of serine/threonine kinases, PAK5, which is a novel target of the Rho GTPases Cdc42 and Rac. The kinase domain and GTPase-binding domain (GBD) of PAK5 are most closely related in sequence to those of mammalian PAK4. Outside of these domains, however, PAK5 is completely different in sequence from any known mammalian proteins. PAK5 does share considerable sequence homology with the Drosophila MBT protein (for “mushroom body tiny”), however, which is thought to play a role in development of cells in Drosophila brain. Interestingly, PAK5 is highly expressed in mammalian brain and is not expressed in most other tissues. We have found that PAK5, like Cdc42, promotes the induction of filopodia. In N1E-115 neuroblastoma cells, expression of PAK5 also triggered the induction of neurite-like processes, and a dominant-negative PAK5 mutant inhibited neurite outgrowth. Expression of activated PAK1 caused no noticeable changes in these cells. An activated mutant of PAK5 had an even more dramatic effect than wild-type PAK5, indicating that the morphologic changes induced by PAK5 are directly related to its kinase activity. Although PAK5 activates the JNK pathway, dominant-negative JNK did not inhibit neurite outgrowth. In contrast, the induction of neurites by PAK5 was abolished by expression of activated RhoA. Previous work has shown that Cdc42 and Rac promote neurite outgrowth by a pathway that is antagonistic to Rho. Our results suggest, therefore, that PAK5 operates downstream to Cdc42 and Rac and antagonizes Rho in the pathway, leading to neurite development. PMID:11756552

Interaction between DISC1 and CHL1 in regulation of neurite outgrowth.

PubMed

Ren, Jun; Zhao, Tian; Xu, Yiliang; Ye, Haihong

2016-10-01

Disrupted-in-schizophrenia 1 (DISC1), a gene susceptible for major mental illnesses, including schizophrenia, plays multiple roles in neural development, including neuronal proliferation, maturation, migration and neurite outgrowth. DISC1 regulates neurite length via interaction with several intracellular proteins, such as NDEL1, FEZ1 and dysbindin. However, the signal transduction mechanism upstream of DISC1 in regulating neurite outgrowth remains to be elucidated. Here we show that DISC1 interacts with the intracellular domain of close homolog of L1 (CHL1), a member of the L1 family of neural cell adhesion molecules. DISC1 and CHL1 proteins co-localize in growth cones of cortical neurons. Moreover, in neurite outgrowth assay, CHL1 rescues the inhibitory effect of DISC1 on the initial phase of neurite outgrowth. Considering the fact that CHL1 also plays crucial roles in neural development, and its coding gene is associated with schizophrenia, our findings indicate that DISC1 and CHL1 may engage in physical and functional interaction in neural development, supporting the notion that DISC1 regulates neurite outgrowth with a receptor belonging to the neural cell adhesion molecules, and disruption of such interaction may contribute to increased risk for schizophrenia. Copyright © 2016. Published by Elsevier B.V.

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.

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

PubMed Central

Shi, Haohong; Luo, Xingjing

2016-01-01

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

Cocaine- and amphetamine-regulated transcript facilitates the neurite outgrowth in cortical neurons after oxygen and glucose deprivation through PTN-dependent pathway.

PubMed

Wang, Y; Qiu, B; Liu, J; Zhu, Wei-Guo; Zhu, S

2014-09-26

Cocaine- and amphetamine-regulated transcript (CART) is a neuropeptide that plays neuroprotective roles in cerebral ischemia and reperfusion (I/R) injury in animal models or oxygen and glucose deprivation (OGD) in cultured neurons. Recent data suggest that intranasal CART treatment facilitates neuroregeneration in stroke brain. However, little is known about the effects of post-treatment with CART during the neuronal recovery after OGD and reoxygenation in cultured primary cortical neurons. The present study was to investigate the role of CART treated after OGD injury in neurons. Primary mouse cortical neurons were subjected to OGD and then treated with CART. Our data show that post-treatment with CART reduced the neuronal apoptosis caused by OGD injury. In addition, CART repaired OGD-impaired cortical neurons by increasing the expression of growth-associated protein 43 (GAP43), which promotes neurite outgrowth. This effect depends on pleiotrophin (PTN) as siRNA-mediated PTN knockdown totally abolished the increase in CART-stimulated GAP43 protein levels. In summary, our findings demonstrate that CART repairs the neuronal injury after OGD by facilitating neurite outgrowth through PTN-dependent pathway. The role for CART in neurite outgrowth makes it a new potential therapeutic agent for the treatment of neurodegenerative diseases. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Electrospun fiber surface nanotopography influences astrocyte-mediated neurite outgrowth.

PubMed

Johnson, Christopher D; D'Amato, Anthony R; Puhl, Devan L; Wich, Douglas M; Vespermann, Amanda; Gilbert, Ryan J

2018-05-15

Aligned, electrospun fiber scaffolds provide topographical guidance for regenerating neurons and glia after central nervous system injury. To date, no study has explored how fiber surface nanotopography affects astrocyte response to fibrous scaffolds. Astrocytes play important roles in the glial scar, the blood brain barrier, and in maintaining homeostasis in the central nervous system. In this study, electrospun poly L-lactic acid fibers were engineered with smooth, pitted, or divoted surface nanotopography. Cortical or spinal cord primary rat astrocytes were cultured on the surfaces for either 1 or 3 days to examine the astrocyte response over time. The results showed that cortical astrocytes were significantly shorter and broader on the pitted and divoted fibers compared to those on smooth fibers. However, spinal cord astrocyte morphology was not significantly altered by the surface features. These findings indicate that astrocytes from unique anatomical locations respond differently to the presence of nanotopography. Western Blot results show that the differences in morphology were not associated with significant changes in GFAP or vinculin in either astrocyte population, suggesting that surface pits and divots do not induce a reactive phenotype in either cortical or spinal cord astrocytes. Finally, astrocytes were co-cultured with dorsal root ganglia to determine how the surfaces affected astrocyte-mediated neurite outgrowth. Astrocytes cultured on the fibers for shorter periods of time (1 day) generally supported longer neurite outgrowth. Pitted and divoted fibers restricted spinal cord astrocyte-mediated neurite outgrowth, while smooth fibers increased 3 day spinal cord astrocyte-mediated neurite outgrowth. In total, fiber surface nanotopography can influence astrocyte elongation and influence the capability of astrocytes to direct neurites. Therefore, fiber surface characteristics should be carefully controlled to optimize astrocyte-mediated axonal

CHLORHEXIDINE INHIBITS L1 CELL ADHESION MOLECULE MEDIATED NEURITE OUTGROWTH IN VITRO

PubMed Central

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

2013-01-01

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

Valproic acid-inducible Arl4D and cytohesin-2/ARNO, acting through the downstream Arf6, regulate neurite outgrowth in N1E-115 cells.

PubMed

Yamauchi, Junji; Miyamoto, Yuki; Torii, Tomohiro; Mizutani, Reiko; Nakamura, Kazuaki; Sanbe, Atsushi; Koide, Hiroshi; Kusakawa, Shinji; Tanoue, Akito

2009-07-15

The mood-stabilizing agent valproic acid (VPA) potently promotes neuronal differentiation. As yet, however, little is known about the underlying molecular mechanism. Here, we show that VPA upregulates cytohesin-2 and mediates neurite outgrowth in N1E-115 neuroblastoma cells. Cytohesin-2 is the guanine-nucleotide exchange factor (GEF) for small GTPases of the Arf family; it regulates many aspects of cellular functions including morphological changes. Treatment with the specific cytohesin family inhibitor SecinH3 or knockdown of cytohesin-2 with its siRNA results in blunted induction of neurite outgrowth in N1E-115 cells. The outgrowth is specifically inhibited by siRNA knockdown of Arf6, but not by that of Arf1. Furthermore, VPA upregulates Arl4D, an Arf-like small GTPase that has recently been identified as the regulator that binds to cytohesin-2. Arl4D knockdown displays an inhibitory effect on neurite outgrowth resulting from VPA, while expression of constitutively active Arl4D induces outgrowth. We also demonstrate that the addition of cell-permeable peptide, coupling the cytohesin-2-binding region of Arl4D into cells, reduces the effect of VPA. Thus, Arl4D is a previously unknown regulator of neurite formation through cytohesin-2 and Arf6, providing another example that the functional interaction of two different small GTPases controls an important cellular function.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flaskos, J., E-mail: flaskos@vet.auth.gr; Nikolaidis, E.; Harris, W.

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, theremore » 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

Gadd45a, the gene induced by the mood stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate paxillin in N1E-115 neuroblastoma cells.

PubMed

Yamauchi, Junji; Miyamoto, Yuki; Murabe, Mayu; Fujiwara, Yoko; Sanbe, Atsushi; Fujita, Yuko; Murase, Shoko; Tanoue, Akito

2007-05-15

Valproic acid (VPA), a mood stabilizer and anticonvulsant, has a variety of neurotrophic functions; however, less is known about how VPA regulates neurite outgrowth. Here, using N1E-115 neuroblastoma cells as the model, we show that VPA upregulates Gadd45a to trigger activation of the downstream JNK cascade controlling neurite outgrowth. VPA induces the phosphorylation of c-Jun N-terminal kinase (JNK) and the substrate paxillin, while VPA induction of neurite outgrowth is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) or a paxillin construct harboring a Ser 178-to-Ala mutation at the JNK phosphorylation. Transfection of Gadd45a, acting through the effector MEKK4, leads to the phosphorylation of the JNK cascade. Conversely, knockdown of Gadd45a with siRNA reduces the effect of VPA. Taken together, these results suggest that upregulation of Gadd45a explains one of the mechanisms whereby VPA induces the neurotrophic effect, providing a new role of Gadd45a in neurite outgrowth.

Activation of transglutaminase 2 by nerve growth factor in differentiating neuroblastoma cells: A role in cell survival and neurite outgrowth.

PubMed

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

2018-02-05

NGF (nerve growth factor) 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 transamidase activity by NGF in retinoic acid-induced differentiating mouse N2a and human SH-SY5Y 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. The role of TG2 in NGF-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 NGF in N2a and SH-SY5Y cells. NGF mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON (Z-ZON-Val-Pro-Leu-OMe; Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-l-valinyl-l-prolinyl-l-leucinmethylester) and R283 (1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride) and by pharmacological inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB) and protein kinase C (PKC), and removal of extracellular Ca 2+ . Fluorescence microscopy demonstrated NGF induced in situ TG2 activity. TG2 inhibition blocked NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth in both cell lines. Together, these results demonstrate that NGF stimulates TG2 transamidase activity via a ERK1/2, PKB and PKC-dependent pathway in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. Furthermore, NGF-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results suggest a novel and important role of TG2 in the cellular functions of NGF. Copyright © 2017 Elsevier B.V. All rights reserved.

The Effects of Hematopoietic Growth Factors on Neurite Outgrowth

PubMed Central

Su, Ye; Cui, Lili; Piao, Chunshu; Li, Bin; Zhao, Li-Ru

2013-01-01

Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) are initially discovered as the essential hematopoietic growth factors regulating bone marrow stem cell proliferation and differentiation, and SCF in combination with G-CSF (SCF+G-CSF) has synergistic effects on bone marrow stem cell mobilization. In this study we have determined the effect of SCF and G-CSF on neurite outgrowth in rat cortical neurons. Using molecular and cellular biology and live cell imaging approaches, we have revealed that receptors for SCF and G-CSF are expressed on the growth core of cortical neurons, and that SCF+G-CSF synergistically enhances neurite extension through PI3K/AKT and NFκB signaling pathways. Moreover, SCF+G-CSF induces much greater NFκB activation, NFκB transcriptional binding and brain-derived neurotrophic factor (BDNF) production than SCF or G-CSF alone. In addition, we have also observed that BDNF, the target gene of NFκB, is required for SCF+G-CSF-induced neurite outgrowth. These data suggest that SCF+G-CSF has synergistic effects to promote neurite growth. This study provides new insights into the contribution of hematopoietic growth factors in neuronal plasticity. PMID:24116056

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.

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 PAC 1 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 PAC 1 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 PAC 1 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 PAC 1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC 1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC 1 receptor. Copyright © 2017 Elsevier Inc. All rights reserved.

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...

The role of chalcones: helichrysetin, xanthohumol, and flavokawin-C in promoting neurite outgrowth in PC12 Adh cells.

PubMed

Phan, Chia-Wei; Sabaratnam, Vikineswary; Yong, Wai-Kuan; Abd Malek, Sri Nurestri

2018-05-01

Chalcones are a group of compounds widely distributed in plant kingdom. The aim of this study was to assess the neurite outgrowth stimulatory activity of selected chalcones, namely helichrysetin, xanthohumol and flavokawin-C. Using adherent rat pheochromocytoma (PC12 Adh) cells, the chalcones were subjected to neurite outgrowth assay and the extracellular nerve growth factor (NGF) levels were determined. Xanthohumol (10 μg/mL) displayed the highest (p < 0.05) percentage of neurite-bearing PC12 Adh cells and the highest (p < 0.05) NGF level in the culture medium of xanthohumol-treated cells. While, helichrysetin induced a moderately high numbers of neurite-bearing cells, flavokawin-C did not stimulate neurite outgrowth. This work supports the potential use of xanthohumol as a potential neuroactive compound to stimulate neurite outgrowth.

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

NASA Astrophysics Data System (ADS)

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

1993-05-01

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

Gold nano-decorated aligned polyurethane nanofibers for enhancement of neurite outgrowth and elongation.

PubMed

Demir, Ulku Selcen; Shahbazi, Reza; Calamak, Semih; Ozturk, Sukru; Gultekinoglu, Merve; Ulubayram, Kezban

2018-06-01

Neurite outgrowth and elongation of neural cells is the most important subject that is considered in nerve tissue engineering. In this regard, aligned nanofibers have taken much attention in terms of providing guidance for newly outgrown neurites. The main objective of this study was to fabricate aligned polyurethane nanofibers by electrospinning process and decorate them with gold nanoparticles to further investigate the synergistic effects of nanotopography, biological nerve growth factor (NGF) and electrical stimulations on neurite outgrowth and elongation of pheochromocytoma (PC-12) model cells. In this regard, smooth and uniform aligned polyurethane nanofibers with the average diameter of 519 ± 56 nm were fabricated and decorated with the gold nanoparticles with the average diameter of ∼50 nm. PC-12 cells were cultured on the various nanofiber surfaces inside the bio-mimetic bioreactor system and exposed either to NGF alone or combination of NGF and electrical stimulation. It was found that 50 ng/mL NGF concentration is an optimal value for the stimulation of neurite outgrowth. After 4 days of culture under 100 mV, 10 ms electrical stimulation in 1 h/day period it was found that the gold nanoparticle decorated aligned polyurethane nanofibers increased the neurite outgrowth and elongation more with the combinational NGF and electrical stimulation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1604-1613, 2018. © 2018 Wiley Periodicals, Inc.

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

PubMed Central

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

2016-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2004-11-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-08-01

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

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.

Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth.

PubMed

Abe, Tomoyuki; Kato, Masayoshi; Miki, Hiroaki; Takenawa, Tadaomi; Endo, Takeshi

2003-01-01

Rho family small GTPases regulate multiple cellular functions through reorganization of the actin cytoskeleton. Among them, Cdc42 and Tc10 induce filopodia or peripheral processes in cultured cells. We have identified a member of the family, designated as RhoT, which is closely related to Tc10. Tc10 was highly expressed in muscular tissues and brain and remarkably induced during differentiation of C2 skeletal muscle cells and neuronal differentiation of PC12 and N1E-115 cells. On the other hand, RhoT was predominantly expressed in heart and uterus and induced during neuronal differentiation of N1E-115 cells. Tc10 exogenously expressed in fibroblasts generated actin-filament-containing peripheral processes longer than the Cdc42-formed filopodia, whereas RhoT produced much longer and thicker processes containing actin filaments. Furthermore, both Tc10 and RhoT induced neurite outgrowth in PC12 and N1E-115 cells, but Cdc42 did not do this by itself. Tc10 and RhoT as well as Cdc42 bound to the N-terminal CRIB-motif-containing portion of N-WASP and activated N-WASP to induce Arp2/3-complex-mediated actin polymerization. The formation of peripheral processes and neurites by Tc10 and RhoT was prevented by the coexpression of dominant-negative mutants of N-WASP. Thus, N-WASP is essential for the process formation and neurite outgrowth induced by Tc10 and RhoT. Neuronal differentiation of PC12 and N1E-115 cells induced by dibutyryl cyclic AMP and by serum starvation, respectively, was prevented by dominant-negative Cdc42, Tc10 and RhoT. Taken together, all these Rho family proteins are required for neuronal differentiation, but they exert their functions differentially in process formation and neurite extension. Consequently, N-WASP activated by these small GTPases mediates neuronal differentiation in addition to its recently identified role in glucose uptake.

Emulsifier for intravenous cyclosporin inhibits neurite outgrowth, causes deficits in rapid axonal transport and leads to structural abnormalities in differentiating N1E.115 neuroblastoma.

PubMed

Brat, D J; Windebank, A J; Brimijoin, S

1992-05-01

The emulsifier for cyclosporin in clinical i.v. formulations, Cremophor EL, has recently come into question as a possible source of neurotoxic side effects in immunosuppressant therapy. To address this issue we tested Cremophor EL and cyclosporin on an in vitro neuronal model, the differentiating N1E.115 neuroblastoma cell. In terms of effects on elaboration of neurites by these cells, Cremophor accounted for nearly all the neurotoxicity of clinically formulated cyclosporin. At a concentration of 0.005% (v/v), Cremophor EL halved the number of cells that extended neurites after 48 hr in serum-free medium. Average neurite length was also reduced substantially. Inhibition of neurite outgrowth first became apparent 24 hr after exposure to Cremophor EL. Neurites that did grow in the presence of Cremophor were disfigured by a series of regularly spaced, gross dilatations (beads) filled with large (0.2-0.5 microns) lipid vesicles. Abnormalities of rapid axonal transport were documented in the beaded neurites by means of video-enhanced contrast, differential interference-contrast microscopy. Velocity of retrograde transport remained normal, but the velocity of anterograde transport and the total bidirectional flux of organelles were both reduced. It seems likely that the inhibition of neurite outgrowth, the swellings of the neurites and the abnormalities of transport are interrelated phenomena.

A robust and reproducible human pluripotent stem cell derived model of neurite outgrowth in a three-dimensional culture system and its application to study neurite inhibition.

PubMed

Clarke, Kirsty E; Tams, Daniel M; Henderson, Andrew P; Roger, Mathilde F; Whiting, Andrew; Przyborski, Stefan A

2017-06-01

The inability of neurites to grow and restore neural connections is common to many neurological disorders, including trauma to the central nervous system and neurodegenerative diseases. Therefore, there is need for a robust and reproducible model of neurite outgrowth, to provide a tool to study the molecular mechanisms that underpin the process of neurite inhibition and to screen molecules that may be able to overcome such inhibition. In this study a novel in vitro pluripotent stem cell based model of human neuritogenesis was developed. This was achieved by incorporating additional technologies, notably a stable synthetic inducer of neural differentiation, and the application of three-dimensional (3D) cell culture techniques. We have evaluated the use of photostable, synthetic retinoid molecules to promote neural differentiation and found that 0.01 μM EC23 was the optimal concentration to promote differentiation and neurite outgrowth from human pluripotent stem cells within our model. We have also developed a methodology to enable quick and accurate quantification of neurite outgrowth derived from such a model. Furthermore, we have obtained significant neurite outgrowth within a 3D culture system enhancing the level of neuritogenesis observed and providing a more physiological microenvironment to investigate the molecular mechanisms that underpin neurite outgrowth and inhibition within the nervous system. We have demonstrated a potential application of our model in co-culture with glioma cells, to recapitulate aspects of the process of neurite inhibition that may also occur in the injured spinal cord. We propose that such a system that can be utilised to investigate the molecular mechanisms that underpin neurite inhibition mediated via glial and neuron interactions. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, Ya-Yun; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; Tseng, Yu-Ting

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-treatedmore » 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

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

PubMed Central

Ishima, T; Iyo, M; Hashimoto, K

2012-01-01

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

Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with α4β1 integrins

PubMed Central

Vogelezang, Mariette; Forster, Ulrike B; Han, Jaewon; Ginsberg, Mark H; ffrench-Constant, Charles

2007-01-01

Background The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for α4β1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the α4 integrin cytoplasmic domain in neurite outgrowth. Results Expression of α4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type α4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal α4-mediated migration in leucocytes requires spatial regulation of α4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this α4(S988D), which mimics phosphorylated α4, did not promote neurite outgrowth. However, α4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable α4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin. Conclusion We establish the importance of the α4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of α4 for extension of neuronal growth cones and migration of non-neural cells. PMID:17603879

Acrylamide and glycidamide impair neurite outgrowth in differentiating N1E.115 neuroblastoma without disturbing rapid bidirectional transport of organelles observed by video microscopy.

PubMed

Brat, D J; Brimijoin, S

1993-06-01

The nature of the pathogenic insult in acrylamide neuropathy is unknown, but axonal transport disturbances are suspected. Using N1E.115 neuroblastoma in vitro, we examined acrylamide and related compounds in terms of general cytotoxicity, ability to block neurite outgrowth, and effects on neurite integrity and fast axonal transport. Acrylamide, glycidamide, and methylene-bis-acrylamide were weakly cytotoxic in a 51Cr-release assay, but only at > or = 10 mM (order of efficacy: methylene-bis-acrylamide > glycidamide > acrylamide). Neurite outgrowth by differentiating cells was inhibited at 100-fold lower concentrations, with similar EC50 values for all three toxicants, i.e., acrylamide, 70 +/- 15 microM; methylene-bis-acrylamide, 92 +/- 31 microM; glycidamide, 120 +/- 30 microM. Only glycidamide (1 mM) caused degeneration of established neurites within a period of 48 h. Video-enhanced contrast differential interference contrast microscopy was used to test the effect of acrylamide and glycidamide on organelle transport in the neurites. In exposures of < or = 48 h at 1 mM, neither toxicant altered bidirectional organelle flux, measured as organelles transported per minute per micrometer of neurite diameter. Anterograde and retrograde organelle speeds were also undisturbed. These results suggest that mechanisms other than direct inhibition of organellar motility are responsible for acrylamide's neurotoxicity in vivo.

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

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

PubMed

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

2015-10-20

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

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.

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

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).

Biofunctionalized peptide-based hydrogels provide permissive scaffolds to attract neurite outgrowth from spiral ganglion neurons.

PubMed

Frick, Claudia; Müller, Marcus; Wank, Ute; Tropitzsch, Anke; Kramer, Benedikt; Senn, Pascal; Rask-Andersen, Helge; Wiesmüller, Karl-Heinz; Löwenheim, Hubert

2017-01-01

Cochlear implants (CI) allow for hearing rehabilitation in patients with sensorineural hearing loss or deafness. Restricted CI performance results from the spatial gap between spiral ganglion neurons and the CI, causing current spread that limits spatially restricted stimulation and impairs frequency resolution. This may be substantially improved by guiding peripheral processes of spiral ganglion neurons towards and onto the CI electrode contacts. An injectable, peptide-based hydrogel was developed which may provide a permissive scaffold to facilitate neurite growth towards the CI. To test hydrogel capacity to attract spiral ganglion neurites, neurite outgrowth was quantified in an in vitro model using a custom-designed hydrogel scaffold and PuraMatrix ® . Neurite attachment to native hydrogels is poor, but significantly improved by incorporation of brain-derived neurotrophic factor (BDNF), covalent coupling of the bioactive laminin epitope IKVAV and the incorporation a full length laminin to hydrogel scaffolds. Incorporation of full length laminin protein into a novel custom-designed biofunctionalized hydrogel (IKVAV-GGG-SIINFEKL) allows for neurite outgrowth into the hydrogel scaffold. The study demonstrates that peptide-based hydrogels can be specifically biofunctionalized to provide a permissive scaffold to attract neurite outgrowth from spiral ganglion neurons. Such biomaterials appear suitable to bridge the spatial gap between neurons and the CI. Copyright © 2016 Elsevier B.V. All rights reserved.

Pea3 transcription factor promotes neurite outgrowth

PubMed Central

Kandemir, Basak; Caglayan, Berrak; Hausott, Barbara; Erdogan, Burcu; Dag, Ugur; Demir, Ozlem; Sogut, Melis S.; Klimaschewski, Lars; Kurnaz, Isil A.

2014-01-01

Pea3 subfamily of E–twenty six transcription factors consist of three major -exhibit branching morphogenesis, the function of Pea3 family in nervous system development and regeneration is only beginning to unfold. In this study, we provide evidence that Pea3 can directs neurite extension and axonal outgrowth in different model systems, and that Serine 90 is important for this function. We have also identified neurofilament-L and neurofilament-M as two putative novel targets for Pea3. PMID:25018694

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.

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

NASA Astrophysics Data System (ADS)

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

2009-02-01

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

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.

Mutation of a putative MAP kinase consensus site regulates NCAM endocytosis and NCAM-dependent neurite outgrowth.

PubMed

Goschzik, Tobias; Cremer, Harold; Gnanapragassam, Vinayaga S; Horstkorte, Rüdiger; Bork, Kaya; Diestel, Simone

2017-07-01

The cytoplasmic domain of the neural cell adhesion molecule NCAM contains several putative serine/threonine phosphorylation sites whose functions are largely unknown. Human NCAM140 (NCAM140) possesses a potential MAP kinase phosphorylation site at threonine (T) 803. The aim of this study was to analyze a possible phosphorylation of NCAM140 by MAP kinases and to identify the functional role of T803. We found that NCAM140 is phosphorylated by the MAP kinase ERK2 in vitro. Exchange of T803 to aspartic acid (D) which mimics constitutive phosphorylation at the respective position resulted in increased endocytosis compared to NCAM140 in neuroblastoma cells and primary neurons. Consistently, NCAM140 endocytosis was inhibited by the MEK inhibitor U0126 in contrast to NCAM140-T803D or NCAM140-T803A endocytosis supporting a role of a potential ERK2 mediated phosphorylation at this site in endocytosis. Furthermore, cells expressing NCAM140-T803D developed significantly shorter neurites than NCAM140 expressing cells indicating that a potential phosphorylation of NCAM by ERK2 also regulates NCAM-dependent neurite outgrowth. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

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

PubMed

Popova, Dina; Jacobsson, Stig O P

2014-04-01

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

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

Guaifenesin derivatives promote neurite outgrowth and protect diabetic mice from neuropathy.

PubMed

Hadimani, Mallinath B; Purohit, Meena K; Vanampally, Chandrashaker; Van der Ploeg, Randy; Arballo, Victor; Morrow, Dwane; Frizzi, Katie E; Calcutt, Nigel A; Fernyhough, Paul; Kotra, Lakshmi P

2013-06-27

In diabetic patients, an early index of peripheral neuropathy is the slowing of conduction velocity in large myelinated neurons and a lack of understanding of the basic pathogenic mechanisms hindered therapeutics development. Racemic (R/S)-guaifenesin (1) was identified as a potent enhancer of neurite outgrowth using an in vitro screen. Its R-enantiomer (R)-1 carried the most biological activity, whereas the S-enantiomer (S)-1 was inactive. Focused structural variations to (R/S)-1 was conducted to identify potentially essential groups for the neurite outgrowth activity. In vivo therapeutic studies indicated that both (R/S)-1 and (R)-1 partially prevented motor nerve conduction velocity slowing in a mouse model of type 1 diabetes. In vitro microsomal assays suggested that compounds (R)-1 and (S)-1 are not metabolized rapidly, and PAMPA assay indicated moderate permeability through the membrane. Findings revealed here could lead to the development of novel drugs for diabetic neuropathy.

Thrombospondin-1 modified bone marrow mesenchymal stem cells (BMSCs) promote neurite outgrowth and functional recovery in rats with spinal cord injury

PubMed Central

Pu, Yujie; Meng, Ke; Gu, Chuanlong; Wang, Linlin; Zhang, Xiaoming

2017-01-01

Stem cell therapies are currently gaining momentum in the treatment of spinal cord injury (SCI). However, unsatisfied intrinsic neurite growth capacity constitutes significant obstacles for injured spinal cord repair and ultimately results in neurological dysfunction. The present study assessed the efficacy of thrombospondin-1 (TSP-1), a neurite outgrowth-promoting molecule, modified bone marrow mesenchymal stem cells (BMSCs) on promoting neurite outgrowth in vitro and in vivo of Oxygen–Glucose Deprivation (OGD) treated motor neurons and SCI rat models. The present results demonstrated that the treatment of BMSCs+TSP-1 could promote the neurite length, neuronal survival, and functional recovery after SCI. Additionally, TSP-1 could activate transforming growth factor-β1 (TGF-β1) then induced the smad2 phosphorylation, and expedited the expression of GAP-43 to promote neurite outgrowth. The present study for the first time demonstrated that BMSCs+TSP-1 could promote neurite outgrowth and functional recovery after SCI partly through the TGF-β1/p-Samd2 pathway. The study provided a novel encouraging evidence for the potential treatment of BMSCs modification with TSP-1 in patients with SCI. PMID:29221205

Low-Intensity Pulsed Ultrasound Enhances Nerve Growth Factor-Induced Neurite Outgrowth through Mechanotransduction-Mediated ERK1/2-CREB-Trx-1 Signaling.

PubMed

Zhao, Lu; Feng, Yi; Hu, Hong; Shi, Aiwei; Zhang, Lei; Wan, Mingxi

2016-12-01

Enhancing the action of nerve growth factor (NGF) is a potential therapeutic approach to neural regeneration. To facilitate neural regeneration, we investigated whether combining low-intensity pulsed ultrasound (LIPUS) and NGF could promote neurite outgrowth, an essential process in neural regeneration. In the present study, PC12 cells were subjected to a combination of LIPUS (1 MHz, 30 or 50 mW/cm 2 , 20% duty cycle and 100-Hz pulse repetition frequency, 10 min every other day) and NGF (50 ng/mL) treatment, and then neurite outgrowth was compared. Our findings indicated that the combined treatment with LIPUS (50 mW/cm 2 ) and NGF (50 ng/mL) promotes neurite outgrowth that is comparable to that achieved by NGF (100 ng/mL) treatment alone. LIPUS significantly increased NGF-induced neurite length, but not neurite branching. These effects were attributed to the enhancing effects of LIPUS on NGF-induced phosphorylation of ERK1/2 and CREB and the expression of thioredoxin (Trx-1). Furthermore, blockage of stretch-activated ion channels with Gd 3+ suppressed the stimulating effects of LIPUS on NGF-induced neurite outgrowth and the downstream signaling activation. Taken together, our findings suggest that LIPUS enhances NGF-induced neurite outgrowth through mechanotransduction-mediated signaling of the ERK1/2-CREB-Trx-1 pathway. The combination of LIPUS and NGF could potentially be used for the treatment of nerve injury and neurodegenerative diseases. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

ACTION OF 50 HZ MAGNETIC FIELDS ON NEURITE OUTGROWTH IN PHEOCHROMOCYTOMA CELLS

EPA Science Inventory

This study tests the capacity of 50-Hz magnetic and electric fields to stimulate neurite outgrowth in PC-12D cells, a cell line which originated from a pheochromocytoma in rat adrenal medulla. he cells were plated on collagen-coated plastic petri dishes and exposed to sinusoidal ...

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

PubMed

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

2013-10-11

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

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.

P2X1 Receptor-Mediated Ca2+ 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.

Effect of medium osmolarity and taurine on neuritic outgrowth from goldfish retinal explants.

PubMed

Cubillán, Lisbeth; Obregón, Francisco; Lima, Lucimey

2009-01-01

Taurine stimulates outgrowth of goldfish retinal explants in a concentration- and time-dependent manner, an effect related to calcium movement and protein phosphorylation. Since taurine is an osmoregulator in the central nervous system, and osmolality might influence regeneration, the purpose of this work was to evaluate the possible effect of hypo-osmolality on basal outgrowth and on the trophic action of the amino acid. Accordingly, goldfish retinal explants obtained after crushing the optic nerve were cultured in iso- and hypo-osmotic medium, the latter achieved by diluting the medium 10% 24 and 72 h after plating. The length and density of the neurites, measured after 5 days in culture, were significantly lower in the hypo- than in the iso-osmotic medium. Taurine stimulated the outgrowth under both conditions, but the percentage of increase was greater in iso-osmotic medium. Taurine concentration, determined by HPLC, did not significantly change in explants. Co-administration of beta-alanine and taurine impaired the trophic effect of taurine to a greater extent in the iso- than in hypo-osmotic medium, indicating a possible differential interaction with the taurine transporter which could be altered by osmotic stress. The exact mechanism of outgrowth regulation by hypotonicity requires further clarification, taking into considering possible modification of the taurine transporter.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marzinke, Mark A.; Clagett-Dame, Margaret, E-mail: dame@biochem.wisc.edu; Pharmaceutical Science Division, University of Wisconsin-Madison, Madison, WI 53705-2222

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, amore » 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.« less

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...

Cadherin genes and evolutionary novelties in the octopus.

PubMed

Wang, Z Yan; Ragsdale, Clifton W

2017-09-01

All animals with large brains must have molecular mechanisms to regulate neuronal process outgrowth and prevent neurite self-entanglement. In vertebrates, two major gene families implicated in these mechanisms are the clustered protocadherins and the atypical cadherins. However, the molecular mechanisms utilized in complex invertebrate brains, such as those of the cephalopods, remain largely unknown. Recently, we identified protocadherins and atypical cadherins in the octopus. The octopus protocadherin expansion shares features with the mammalian clustered protocadherins, including enrichment in neural tissues, clustered head-to-tail orientations in the genome, and a large first exon encoding all cadherin domains. Other octopus cadherins, including a newly-identified cadherin with 77 extracellular cadherin domains, are elevated in the suckers, a striking cephalopod novelty. Future study of these octopus genes may yield insights into the general functions of protocadherins in neural wiring and cadherin-related proteins in complex morphogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

A patterned recombinant human IgM guides neurite outgrowth of CNS neurons

PubMed Central

Xu, Xiaohua; Wittenberg, Nathan J.; Jordan, Luke R.; Kumar, Shailabh; Watzlawik, Jens O.; Warrington, Arthur E.; Oh, Sang-Hyun; Rodriguez, Moses

2013-01-01

Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks. PMID:23881231

Electrical stimulation of schwann cells promotes sustained increases in neurite outgrowth.

PubMed

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

2014-02-01

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

Emodin induces neurite outgrowth through PI3K/Akt/GSK-3β-mediated signaling pathways in Neuro2a cells.

PubMed

Park, Shin-Ji; Jin, Mei Ling; An, Hyun-Kyu; Kim, Kyoung-Sook; Ko, Min Jung; Kim, Cheol Min; Choi, Young Whan; Lee, Young-Choon

2015-02-19

In this study, a neurite outgrowth-inducing substance was isolated from the ethylacetate extract of the Polygonum multiflorum roots and identified as emodin by gas-liquid chromatography-mass spectrometry and (1)H NMR and (13)C NMR. Emodin displayed remarkable neurite outgrowth-inducing activity in Neuro2a cells, as demonstrated by morphological changes and immunocytochemistry for class III β-tubulin. Emodin exhibited a stronger neutrophic activity than retinoic acid (RA) known as inducer of neurite outgrowth in Neuro2a cells. Emodin treatment resulted in marked increases in phosphorylation of Akt a direct downstream signaling molecule of phosphatidylinositol 3-kinase (PI3K), but upstream of glycogen synthase kinase-3β (GSK-3β) and cAMP response element-binding protein (CREB). These augmentations and neurite-bearing cells induced by emodin were remarkably reduced by the addition of PI3K inhibitor LY294002. These results demonstrate that emodin induces neuronal differentiation of Neuro2a cells via PI3K/Akt/GSK-3β pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Stargazin-related protein γ7 is associated with signalling endosomes in superior cervical ganglion neurons and modulates neurite outgrowth

PubMed Central

Waithe, Dominic; Ferron, Laurent; Dolphin, Annette C.

2011-01-01

The role(s) of the newly discovered stargazin-like γ-subunit proteins remains unclear; although they are now widely accepted to be transmembrane AMPA receptor regulatory proteins (TARPs), rather than Ca2+ channel subunits, it is possible that they have more general roles in trafficking within neurons. We previously found that γ7 subunit is associated with vesicles when it is expressed in neurons and other cells. Here, we show that γ7 is present mainly in retrogradely transported organelles in sympathetic neurons, where it colocalises with TrkA–YFP, and with the early endosome marker EEA1, suggesting that γ7 localises to signalling endosomes. It was not found to colocalise with markers of the endoplasmic reticulum, mitochondria, lysosomes or late endosomes. Furthermore, knockdown of endogenous γ7 by short hairpin RNA transfection into sympathetic neurons reduced neurite outgrowth. The same was true in the PC12 neuronal cell line, where neurite outgrowth was restored by overexpression of human γ7. These findings open the possibility that γ7 has an essential trafficking role in relation to neurite outgrowth as a component of endosomes involved in neurite extension and growth cone remodelling. PMID:21610096

Dietary uridine-5'-monophosphate supplementation increases potassium-evoked dopamine release and promotes neurite outgrowth in aged rats.

PubMed

Wang, Lei; Pooler, Amy M; Albrecht, Meredith A; Wurtman, Richard J

2005-01-01

Membrane phospholipids like phosphatidylcholine (PC) are required for cellular growth and repair, and specifically for synaptic function. PC synthesis is controlled by cellular levels of its precursor, cytidine-5'-diphosphate choline (CDP-choline), which is produced from cytidine triphosphate (CTP) and phosphocholine. In rat PC12 cells exogenous uridine was shown to elevate intracellular CDP-choline levels, by promoting the synthesis of uridine triphosphate (UTP), which was partly converted to CTP. In such cells uridine also enhanced the neurite outgrowth produced by nerve growth factor (NGF). The present study assessed the effect of dietary supplementation with uridine-5'-monophosphate disodium (UMP-2Na+, an additive in infant milk formulas) on striatal dopamine (DA) release in aged rats. Male Fischer 344 rats consumed either a control diet or one fortified with 2.5% UMP for 6 wk, ad libitum. In vivo microdialysis was then used to measure spontaneous and potassium (K+)-evoked DA release in the right striatum. Potassium (K+)-evoked DA release was significantly greater among UMP-treated rats, i.e., 341+/-21% of basal levels vs. 283+/-9% of basal levels in control rats (p<0.05); basal DA release was unchanged. In general, each animal's K+-evoked DA release correlated with its striatal DA content, measured postmortem. The levels of neurofilament-70 and neurofilament-M proteins, biomarkers of neurite outgrowth, increased to 182+/-25% (p<0.05) and 221+/-34% (p<0.01) of control values, respectively, with UMP consumption. Hence, UMP treatment not only enhances membrane phosphatide production but also can modulate two membrane-dependent processes, neurotransmitter release and neurite outgrowth, in vivo.

Actin Waves Do Not Boost Neurite Outgrowth in the Early Stages of Neuron Maturation

PubMed Central

Mortal, Simone; Iseppon, Federico; Perissinotto, Andrea; D'Este, Elisa; Cojoc, Dan; Napolitano, Luisa M. R.; Torre, Vincent

2017-01-01

During neurite development, Actin Waves (AWs) emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC). Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration. PMID:29326552

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

PubMed Central

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

2014-01-01

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

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

PubMed Central

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

2012-01-01

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

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

PubMed Central

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

2015-01-01

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

Chemical Constituents from Hericium erinaceus Promote Neuronal Survival and Potentiate Neurite Outgrowth via the TrkA/Erk1/2 Pathway.

PubMed

Zhang, Cheng-Chen; Cao, Chen-Yu; Kubo, Miwa; Harada, Kenichi; Yan, Xi-Tao; Fukuyama, Yoshiyasu; Gao, Jin-Ming

2017-07-30

Hericium erinaceus is a culinary-medicinal mushroom used traditionally in Eastern Asia to improve memory. In this work, we investigated the neuroprotective and neuritogenic effects of the secondary metabolites isolated from the MeOH extract of cultured mycelium of H. erinaceus and the primary mechanisms involved. One new dihydropyridine compound ( 6 ) and one new natural product ( 2 ) together with five known compounds ( 1 , 3 - 5 , 7 ) were obtained and their structures were elucidated by spectroscopic analysis, including 2D NMR and HRMS. The cell-based screening for bioactivity showed that 4-chloro-3,5-dimethoxybenzoic methyl ester ( 1 ) and a cyathane diterpenoid, erincine A ( 3 ), not only potentiated NGF-induced neurite outgrowth but also protected neuronally-differentiated cells against deprivation of NGF in PC12 pheochromocytoma cells. Additionally, compound 3 induced neuritogenesis in primary rat cortex neurons. Furthermore, our results revealed that TrkA-mediated and Erk1/2-dependant pathways could be involved in 1 and 3 -promoted NGF-induced neurite outgrowth in PC12 cells.

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...

IGF-1 and BDNF promote chick bulbospinal neurite outgrowth in vitro.

PubMed

Salie, Rishard; Steeves, John D

2005-11-01

Injured neurons in the CNS do not experience significant functional regeneration and so spinal cord insult often results in permanently compromised locomotor ability. The capability of a severed axon to re-grow is thought to depend on numerous factors, one of which is the decreased availability of neurotrophic factors. Application of trophic factors to axotomized neurons has been shown to enhance survival and neurite outgrowth. Although brainstem-spinal connections play a pivotal role in motor dysfunction after spinal cord injury, relatively little is known about the trophic sensitivity of these populations. This study explores the response of bulbospinal populations to various trophic factors. Several growth factors were initially examined for potential trophic effects on the projection neurons of the brainstem. Brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) significantly enhance mean process length in both the vestibulospinal neurons and spinal projection neurons from the raphe nuclei. Nerve growth factor (NGF), neurotrophin-4 (NT-4) and glial derived neurotrophic factor (GDNF) did not effect process outgrowth in vestibulospinal neurons. At the developmental stages used in this study, it was determined that receptors for BDNF and IGF-1 were present both on bulbospinal neurons and on surrounding cells with a non-neuronal morphology.

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

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

PubMed

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

2015-01-01

Hericium erinaceus has been reported to have a wide range of medicinal properties such as stimulation of neurite outgrowth, promotion of functional recovery of axonotmetic peroneal nerve injury, antioxidant, antihypertensive, and antidiabetic properties. In recent years, the green synthesis of gold nanoparticles (AuNPs) has attracted intense interest due to the potential use in biomedical applications. The aim of this study was to investigate the effects of AuNPs from aqueous extract of H. erinaceus on neurite outgrowth of rat pheochromocytoma (PC-12) cells. The formation of AuNPs was characterized by UV-visible spectrum, energy dispersive X-ray (EDX), field-emission scanning electron microscope (FESEM), transmission electron microscopy (TEM), particle size distribution, and Fourier transform-infrared spectroscopy (FTIR). Furthermore, the neurite extension study of synthesized AuNPs was evaluated by in vitro assay. The AuNPs exhibited maximum absorbance between 510 and 600 nm in UV-visible spectrum. FESEM and TEM images showed the existence of nanoparticles with sizes of 20-40 nm. FTIR measurements were carried out to identify the possible biomolecules responsible for capping and efficient stabilization of the nanoparticles. The purity and the crystalline properties were confirmed by EDX diffraction analysis, which showed strong signals with energy peaks in the range of 2-2.4 keV, indicating the existence of gold atoms. The synthesized AuNPs showed significant neurite extension on PC-12 cells. Nerve growth factor 50 ng/mL was used as a positive control. Treatment with different concentrations (nanograms) of AuNPs resulted in neuronal differentiation and neuronal elongation. AuNPs induced maximum neurite outgrowth of 13% at 600 ng/mL concentration. In this study, the AuNPs synthesis was achieved by a simple, low-cost, and rapid bioreduction approach. AuNPs were shown to have potential neuronal differentiation and stimulated neurite outgrowth. The water

LIF is more potent than BDNF in promoting neurite outgrowth of mammalian auditory neurons in vitro.

PubMed

Gillespie, L N; Clark, G M; Bartlett, P F; Marzella, P L

2001-02-12

Neurotrophic factors are known to play a crucial role in the elongation and guidance of auditory nerve fibres to their targets within the organ of Corti. Maintenance of these neural connections following deafness would clearly influence the efficacy of therapies for hearing recovery. The growth factors leukaemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF) and transforming growth factor-beta 5 (TGF-beta5) were tested for their efficacy in promoting neurite outgrowth from dissociated cultures of early postnatal rat auditory neurons. Our results indicate that while BDNF enhances neurite outgrowth in a strong fashion, LIF is more potent; moreover, the combined administration of both factors has even greater neuritogenic capacities. TGF-beta5, although neurotrophic, has no neuritogenic activity on cultured auditory neurons. LIF and BDNF may therefore be potential candidates when developing pharmacological therapies for hearing recovery.

IL-10 Promotes Neurite Outgrowth and Synapse Formation in Cultured Cortical Neurons after the Oxygen-Glucose Deprivation via JAK1/STAT3 Pathway.

PubMed

Chen, Hongbin; Lin, Wei; Zhang, Yixian; Lin, Longzai; Chen, Jianhao; Zeng, Yongping; Zheng, Mouwei; Zhuang, Zezhong; Du, Houwei; Chen, Ronghua; Liu, Nan

2016-07-26

As a classic immunoregulatory and anti-inflammatory cytokine, interleukin-10 (IL-10) provides neuroprotection in cerebral ischemia in vivo or oxygen-glucose deprivation (OGD)-induced injury in vitro. However, it remains blurred whether IL-10 promotes neurite outgrowth and synapse formation in cultured primary cortical neurons after OGD injury. In order to evaluate its effect on neuronal apoptosis, neurite outgrowth and synapse formation, we administered IL-10 or IL-10 neutralizing antibody (IL-10NA) to cultured rat primary cortical neurons after OGD injury. We found that IL-10 treatment activated the Janus kinase 1 (JAK1)/signal transducers and activators of transcription 3 (STAT3) signaling pathway. Moreover, IL-10 attenuated OGD-induced neuronal apoptosis by down-regulating the Bax expression and up-regulating the Bcl-2 expression, facilitated neurite outgrowth by increasing the expression of Netrin-1, and promoted synapse formation in cultured primary cortical neurons after OGD injury. These effects were partly abolished by JAK1 inhibitor GLPG0634. Contrarily, IL-10NA produced opposite effects on the cultured cortical neurons after OGD injury. Taken together, our findings suggest that IL-10 not only attenuates neuronal apoptosis, but also promotes neurite outgrowth and synapse formation via the JAK1/STAT3 signaling pathway in cultured primary cortical neurons after OGD injury.

Atorvastatin enhances neurite outgrowth in cortical neurons in vitro via up-regulating the Akt/mTOR and Akt/GSK-3β signaling pathways

PubMed Central

Jin, Ying; Sui, Hai-juan; Dong, Yan; Ding, Qi; Qu, Wen-hui; Yu, Sheng-xue; Jin, Ying-xin

2012-01-01

Aim: To investigate whether atorvastatin can promote formation of neurites in cultured cortical neurons and the signaling mechanisms responsible for this effect. Methods: Cultured rat cerebral cortical neurons were incubated with atorvastatin (0.05–10 μmol/L) for various lengths of time. For pharmacological experiments, inhibitors were added 30 min prior to addition of atorvastatin. Control cultures received a similar amount of DMSO. Following the treatment period, phase-contrast digital images were taken. Digital images of neurons were analyzed for total neurite branch length (TNBL), neurite number, terminal branch number, and soma area by SPOT Advanced Imaging software. After incubation with atorvastatin for 48 h, the levels of phosphorylated 3-phosphoinoside-dependent protein kinase-1 (PDK1), phospho-Akt, phosphorylated mammalian target of rapamycin (mTOR), phosphorylated 4E-binding protein 1 (4E-BP1), p70S6 kinase (p70S6K), and glycogen synthase kinase-3β (GSK-3β) in the cortical neurons were evaluated using Western blotting analyses. Results: Atorvastatin (0.05–10 μmol/L) resulted in dose-dependent increase in neurite number and length in these neurons. Pretreatment of the cortical neurons with phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 (30 μmol/L) and wortmannin (5 μmol/L), Akt inhibitor tricribine (1 μmol/L) or mTOR inhibitor rapamycin (100 nmol/L) blocked the atorvastatin-induced increase in neurite outgrowth, suggesting that atorvastatin promoted neurite outgrowth via activating the PI3K/Akt/mTOR signaling pathway. Atorvastatin (10 μmol/L) significantly increased the levels of phosphorylated PDK1, Akt and mTOR in the cortical neurons, which were prevented by LY294002 (30 μmol/L). Moreover, atorvastatin (10 μmol/L) stimulated the phosphorylation of 4E-BP1 and p70S6K, the substrates of mTOR, in the cortical neurons. In addition, atorvastatin (10 μmol/L) significantly increased the phosphorylated GSK-3β level in the cortical

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

PubMed

Zhou, Xiaohua; Tai, Akihiro; Yamamoto, Itaru

2003-03-01

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

Chemical Constituents from Hericium erinaceus Promote Neuronal Survival and Potentiate Neurite Outgrowth via the TrkA/Erk1/2 Pathway

PubMed Central

Cao, Chen-Yu; Kubo, Miwa; Harada, Kenichi; Yan, Xi-Tao; Fukuyama, Yoshiyasu; Gao, Jin-Ming

2017-01-01

Hericium erinaceus is a culinary-medicinal mushroom used traditionally in Eastern Asia to improve memory. In this work, we investigated the neuroprotective and neuritogenic effects of the secondary metabolites isolated from the MeOH extract of cultured mycelium of H. erinaceus and the primary mechanisms involved. One new dihydropyridine compound (6) and one new natural product (2) together with five known compounds (1,3–5,7) were obtained and their structures were elucidated by spectroscopic analysis, including 2D NMR and HRMS. The cell-based screening for bioactivity showed that 4-chloro-3,5-dimethoxybenzoic methyl ester (1) and a cyathane diterpenoid, erincine A (3), not only potentiated NGF-induced neurite outgrowth but also protected neuronally-differentiated cells against deprivation of NGF in PC12 pheochromocytoma cells. Additionally, compound 3 induced neuritogenesis in primary rat cortex neurons. Furthermore, our results revealed that TrkA-mediated and Erk1/2-dependant pathways could be involved in 1 and 3-promoted NGF-induced neurite outgrowth in PC12 cells. PMID:28758954

IL-10 Promotes Neurite Outgrowth and Synapse Formation in Cultured Cortical Neurons after the Oxygen-Glucose Deprivation via JAK1/STAT3 Pathway

PubMed Central

Chen, Hongbin; Lin, Wei; Zhang, Yixian; Lin, Longzai; Chen, Jianhao; Zeng, Yongping; Zheng, Mouwei; Zhuang, Zezhong; Du, Houwei; Chen, Ronghua; Liu, Nan

2016-01-01

As a classic immunoregulatory and anti-inflammatory cytokine, interleukin-10 (IL-10) provides neuroprotection in cerebral ischemia in vivo or oxygen-glucose deprivation (OGD)-induced injury in vitro. However, it remains blurred whether IL-10 promotes neurite outgrowth and synapse formation in cultured primary cortical neurons after OGD injury. In order to evaluate its effect on neuronal apoptosis, neurite outgrowth and synapse formation, we administered IL-10 or IL-10 neutralizing antibody (IL-10NA) to cultured rat primary cortical neurons after OGD injury. We found that IL-10 treatment activated the Janus kinase 1 (JAK1)/signal transducers and activators of transcription 3 (STAT3) signaling pathway. Moreover, IL-10 attenuated OGD-induced neuronal apoptosis by down-regulating the Bax expression and up-regulating the Bcl-2 expression, facilitated neurite outgrowth by increasing the expression of Netrin-1, and promoted synapse formation in cultured primary cortical neurons after OGD injury. These effects were partly abolished by JAK1 inhibitor GLPG0634. Contrarily, IL-10NA produced opposite effects on the cultured cortical neurons after OGD injury. Taken together, our findings suggest that IL-10 not only attenuates neuronal apoptosis, but also promotes neurite outgrowth and synapse formation via the JAK1/STAT3 signaling pathway in cultured primary cortical neurons after OGD injury. PMID:27456198

Enhanced differentiation of neural stem cells to neurons and promotion of neurite outgrowth by oxygen-glucose deprivation.

PubMed

Wang, Qin; Yang, Lin; Wang, Yaping

2015-06-01

Stroke has become the leading cause of mortality worldwide. Hypoxic or ischemic insults are crucial factors mediating the neural damage in the brain tissue of stroke patients. Neural stem cells (NSCs) have been recognized as a promising tool for the treatment of ischemic stroke and other neurodegenerative diseases due to their inducible pluripotency. In this study, we aim to mimick the cerebral hypoxic-ischemic injury in vitro using oxygen-glucose deprivation (OGD) strategy, and evaluate the effects of OGD on the NSC's neural differentiation, as well as the differentiated neurite outgrowth. Our data showed that NSCs under the short-term 2h OGD treatment are able to maintain cell viability and the capability to form neurospheres. Importantly, this moderate OGD treatment promotes NSC differentiation to neurons and enhances the performance of the mature neuronal networks, accompanying increased neurite outgrowth of differentiated neurons. However, long-term 6h and 8h OGD exposures in NSCs lead to decreased cell survival, reduced differentiation and diminished NSC-derived neurite outgrowth. The expressions of neuron-specific microtubule-associated protein 2 (MAP-2) and growth associated protein 43 (GAP-43) are increased by short-term OGD treatments but suppressed by long-term OGD. Overall, our results demonstrate that short-term OGD exposure in vitro induces differentiation of NSCs while maintaining their proliferation and survival, providing valuable insights of adopting NSC-based therapy for ischemic stroke and other neurodegenerative disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2011-08-01

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

The application of magnets directs the orientation of neurite outgrowth in cultured human neuronal cells.

PubMed

Kim, Seungchan; Im, Woo-Seok; Kang, Lami; Lee, Soon-Tae; Chu, Kon; Kim, Byoung In

2008-09-15

Electric and magnetic fields have been known to influence cellular behavior. In the present study, we hypothesized that the application of static magnetic fields to neurons will cause neurites to grow in a specific direction. In cultured human neuronal SH-SY5Y cells or PC12 cells, neurite outgrowth was induced by forskolin, retinoic acid, or nerve growth factor (NGF). We applied static magnetic fields to the neurons and analyzed the direction and morphology of newly formed neuronal processes. In the presence of the magnetic field, neurites grew in a direction perpendicular to the direction of the magnetic field, as revealed by the higher orientation index of neurites grown under the magnetic field compared to that of the neurites grown in the absence of the magnetic field. The neurites parallel to the magnetic field appeared to be dystrophic, beaded or thickened, suggesting that they would hinder further elongation processes. The co-localized areas of microtubules and actin filaments were arranged into the vertical axis to the magnetic field, while the levels of neurofilament and synaptotagmin were not altered. Our results suggest that the application of magnetic field can be used to modulate the orientation and direction of neurite formation in cultured human neuronal cells.

Microelectrode array-induced neuronal alignment directs neurite outgrowth: analysis using a fast Fourier transform (FFT).

PubMed

Radotić, Viktorija; Braeken, Dries; Kovačić, Damir

2017-12-01

Many studies have shown that the topography of the substrate on which neurons are cultured can promote neuronal adhesion and guide neurite outgrowth in the same direction as the underlying topography. To investigate this effect, isotropic substrate-complementary metal-oxide-semiconductor (CMOS) chips were used as one example of microelectrode arrays (MEAs) for directing neurite growth of spiral ganglion neurons. Neurons were isolated from 5 to 7-day-old rat pups, cultured 1 day in vitro (DIV) and 4 DIV, and then fixed with 4% paraformaldehyde. For analysis of neurite alignment and orientation, fast Fourier transformation (FFT) was used. Results revealed that on the micro-patterned surface of a CMOS chip, neurons orient their neurites along three directional axes at 30, 90, and 150° and that neurites aligned in straight lines between adjacent pillars and mostly followed a single direction while occasionally branching perpendicularly. We conclude that the CMOS substrate guides neurites towards electrodes by means of their structured pillar organization and can produce electrical stimulation of aligned neurons as well as monitoring their neural activities once neurites are in the vicinity of electrodes. These findings are of particular interest for neural tissue engineering with the ultimate goal of developing a new generation of MEA essential for improved electrical stimulation of auditory neurons.

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...

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.

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...

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

cAMP-induced activation of protein kinase A and p190B RhoGAP mediates down-regulation of TC10 activity at the plasma membrane and neurite outgrowth.

PubMed

Koinuma, Shingo; Takeuchi, Kohei; Wada, Naoyuki; Nakamura, Takeshi

2017-11-01

Cyclic AMP plays a pivotal role in neurite growth. During outgrowth, a trafficking system supplies membrane at growth cones. However, the cAMP-induced signaling leading to the regulation of membrane trafficking remains unknown. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking. Recent studies have shown a role of TC10 in neurite growth in NGF-treated PC12 cells. Here, we investigated a mechanical linkage between cAMP and TC10 in neuritogenesis. Plasmalemmal TC10 activity decreased abruptly after cAMP addition in neuronal cells. TC10 was locally inactivated at extending neurite tips in cAMP-treated PC12 cells. TC10 depletion led to a decrease in cAMP-induced neurite outgrowth. Constitutively active TC10 could not rescue this growth reduction, supporting our model for a role of GTP hydrolysis of TC10 in neuritogenesis by accelerating vesicle fusion. The cAMP-induced TC10 inactivation was mediated by PKA. Considering cAMP-induced RhoA inactivation, we found that p190B, but not p190A, mediated inactivation of TC10 and RhoA. Upon cAMP treatment, p190B was recruited to the plasma membrane. STEF depletion and Rac1-N17 expression reduced cAMP-induced TC10 inactivation. Together, the PKA-STEF-Rac1-p190B pathway leading to inactivation of TC10 and RhoA at the plasma membrane plays an important role in cAMP-induced neurite outgrowth. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Enhanced total neurite outgrowth and secondary branching in dorsal root ganglion neurons elicited by low intensity pulsed ultrasound.

PubMed

Ventre, Daniel; Puzan, Marissa; Ashbolt, Emily; Koppes, Abigail

2018-04-17

Despite the prevalence of peripheral nerve injuries (PNI), challenges remain in restoring full functionality to those afflicted. For recovery to occur, axons must extend across the injury site to connect with distal targets, where injury gap size is a critical factor in the probability of restoration of function. Current clinical therapies often achieve limited neural regeneration, motivating the development of new therapeutic interventions such as biophysical stimulation. To investigate the potential for low intensity, pulsed ultrasonic simulation (LIPUS) to impact peripheral nerve regeneration, primary neonatal rat dorsal root ganglion neurons were examined in vitro in response to ultrasound (US). Dissociated neurons were stimulated with varied acoustic power (low, medium, high) and their morphometrics, including total outgrowth, branching, and length, were analyzed acutely after 18 h of growth. Results show US increases total neurite outgrowth by 2.83-fold compared to unstimulated controls at the highest power. Neurite branching at medium and high-power US increased approximately 2-fold compared to controls, while low stimulation exhibited more muted trends. Neurite branching is also impacted by US, with medium and high power eliciting the highest branching, of approximately 2-fold compared to low power and unstimulated controls. These results demonstrate that US stimulation of DRG neurons in vitro impacts neurite morphology and enhances total extension, indicating the potential for advancing and understanding driving mechanisms of ultrasonic therapies for peripheral nerve regeneration.

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...

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

PubMed Central

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

2011-01-01

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

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

PubMed Central

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

2013-01-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Chih-Hao; Neurosurgery, Department of Surgery, Kaohsiung Veterans General Hospital, Taiwan, ROC; Department of Biomedical Engineering, I-Shou University, Taiwan, ROC

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 porousmore » 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.« less

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

PubMed Central

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

2016-01-01

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

Salicin from Willow Bark can Modulate Neurite Outgrowth in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Wölfle, Ute; Haarhaus, Birgit; Kersten, Astrid; Fiebich, Bernd; Hug, Martin J; Schempp, Christoph M

2015-10-01

Salicin from willow bark has been used throughout centuries in China and Europe for the treatment of pain, headache, and inflammatory conditions. Recently, it could be demonstrated that salicin binds and activates the bitter taste receptor TAS2R16. Studies on rodent tissues showed the general expression of bitter taste receptors (TAS2Rs) in rodent brain. Here, we demonstrate the expression of hTAS2R16 in human neuronal tissues and the neuroblastoma cell line SH-SY5Y. The functionality was analyzed in the neuroblastoma cell line SH-SY5Y after stimulation with salicin, a known TAS2R16 agonist. In this setting salicin induced in SH-SY5Y cells phosphorylation of ERK and CREB, the key transcription factor of neuronal differentiation. PD98059, an inhibitor of the ERK pathway, as well as probenecid, a TAS2R16 antagonist, inhibited receptor phosphorylation as well as neurite outgrowth. These data show that salicin might modulate neurite outgrowth by bitter taste receptor activation. Copyright © 2015 John Wiley & Sons, Ltd.

Guidance of dorsal root ganglion neurites and Schwann cells by isolated Schwann cell topography on poly(dimethyl siloxane) conduits and films

NASA Astrophysics Data System (ADS)

Richardson, J. A.; Rementer, C. W.; Bruder, Jan M.; Hoffman-Kim, D.

2011-08-01

Biomimetic replicas of cellular topography have been utilized to direct neurite outgrowth. Here, we cultured postnatal rat dorsal root ganglion (DRG) explants in the presence of Schwann cell (SC) topography to determine the influence of SC topography on neurite outgrowth. Four distinct poly(dimethyl siloxane) conduits were fabricated within which DRG explants were cultured. To determine the contribution of SC topographical features to neurite guidance, the extent of neurite outgrowth into unpatterned conduits, conduits with randomly oriented SC replicas, and conduits with SC replicas parallel or perpendicular to the conduit long axis was measured. Neurite directionality and outgrowth from DRG were also quantified on two-dimensional SC replicas with orientations corresponding to the four conduit conditions. Additionally, live SC migration and neurite extension from DRG on SC replicas were examined as a first step toward quantification of the interactions between live SC and navigating neurites on SC replicas. DRG neurite outgrowth and morphology within conduits and on two-dimensional SC replicas were directed by the underlying SC topographical features. Maximal neurite outgrowth and alignment to the underlying features were observed into parallel conduits and on parallel two-dimensional substrates, whereas the least extent of outgrowth was observed into perpendicular conduits and on perpendicular two-dimensional replica conditions. Additionally, neurites on perpendicular conditions turned to extend along the direction of underlying SC topography. Neurite outgrowth exceeded SC migration in the direction of the underlying anisotropic SC replica after two days in culture. This finding confirms the critical role that SC have in guiding neurite outgrowth and suggests that the mechanism of neurite alignment to SC replicas depends on direct contact with cellular topography. These results suggest that SC topographical replicas may be used to direct and optimize neurite

Concentration dependent survival and neural differentiation of murine embryonic stem cells cultured on polyethylene glycol dimethacrylate hydrogels possessing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-Asp-Lle.

PubMed

Lim, Hyun Ju; Mosley, Matthew C; Kurosu, Yuki; Smith Callahan, Laura A

2017-07-01

N-cadherin cell-cell signaling plays a key role in the structure and function of the nervous system. However, few studies have incorporated bioactive signaling from n-cadherin into tissue engineering matrices. The present study uses a continuous gradient approach in polyethylene glycol dimethacrylate hydrogels to identify concentration dependent effects of n-cadherin peptide, His-Ala-Val-Asp-Lle (HAVDI), on murine embryonic stem cell survival and neural differentiation. The n-cadherin peptide was found to affect the expression of pluripotency marker, alkaline phosphatase, in murine embryonic stem cells cultured on n-cadherin peptide containing hydrogels in a concentration dependent manner. Increasing n-cadherin peptide concentrations in the hydrogels elicited a biphasic response in neurite extension length and mRNA expression of neural differentiation marker, neuron-specific class III β-tubulin, in murine embryonic stem cells cultured on the hydrogels. High concentrations of n-cadherin peptide in the hydrogels were found to increase the expression of apoptotic marker, caspase 3/7, in murine embryonic stem cells compared to that of murine embryonic stem cell cultures on hydrogels containing lower concentrations of n-cadherin peptide. Increasing the n-cadherin peptide concentration in the hydrogels facilitated greater survival of murine embryonic stem cells exposed to increasing oxidative stress caused by hydrogen peroxide exposure. The combinatorial approach presented in this work demonstrates concentration dependent effects of n-cadherin signaling on mouse embryonic stem cell behavior, underscoring the need for the greater use of systematic approaches in tissue engineering matrix design in order to understand and optimize bioactive signaling in the matrix for tissue formation. Single cell encapsulation is common in tissue engineering matrices. This eliminates cellular access to cell-cell signaling. N-cadherin, a cell-cell signaling molecule, plays a vital role in

Comparison of lawsone contents among Lawsonia inermis plant parts and neurite outgrowth accelerators from branches.

PubMed

Oda, Yoshimi; Nakashima, Souichi; Kondo, Erina; Nakamura, Seikou; Yano, Mamiko; Kubota, Chisa; Masumoto, Yusuke; Hirao, Minami; Ogawa, Yuki; Matsuda, Hisashi

2018-05-17

Lawsonia inermis L. (Lythraceae) is cultivated in many countries, including Japan, China, India and Egypt. Its leaves are well known as hair dye and exhibit antibacterial, anticancer, antifungal and anticonvulsant activities. However, there are few reports on its branches. 2-Hydroxy-1,4-naphthoquinone (lawsone) is a characteristic compound in its leaves. However, it remains to be clarified whether lawsone is present in other plant parts or not. In this study, we measured lawsone contents in the extracts of L. inermis flowers, leaves and branches by HPLC with tandem mass spectrometry. The extracts of L. inermis flowers, leaves and branches contained 116.9, 486.2 and 5.4 μg/g lawsone, respectively. Lawsone content was much lower in branches than the other plant parts. Next, in order to identify the biological constituents in the branches, we isolated nine known compounds and examined their effects on neurite outgrowth in PC12 cells. Among the constituents isolated, 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol (1) and quercetin 7-O-β-D-glucopyranoside (8) showed accelerative effects on neurite outgrowth in PC12 cells.

Intracellular chloride ion concentration in differentiating neuronal cell and its role in growing neurite

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakajima, Ken-ichi; Marunaka, Yoshinori; Department of Bio-Ionomics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kyoto 602-8566

Chloride ion (Cl{sup −}) is one of the most abundant anions in our body. Increasing evidence suggests that Cl{sup −} plays fundamental roles in various cellular functions. We have previously reported that electroneutral cation-chloride cotransporters, such as Na{sup +}-K{sup +}-2Cl{sup −} cotransporter 1 (NKCC1) and K{sup +}-Cl{sup −} cotransporter 1 (KCC1), are involved in neurite outgrowth during neuronal differentiation. In the present study, we studied if there is correlation between intracellular Cl{sup −} concentrations ([Cl{sup −}]{sub i}) and the length of growing neurites. We measured [Cl{sup −}]{sub i} in the cell body and growing neurite tips using halide-sensitive fluorescent dyemore » N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), revealing that [Cl{sup −}]{sub i} in the tip of growing neurite was higher than that in cell body in a single cell. Importantly, there was a significant positive correlation between the length of growing neurite and [Cl{sup −}]{sub i} in neurite tip. Bumtanide (BMT), an inhibitor of NKCC1, significantly inhibited neurite outgrowth and decreased [Cl{sup −}]{sub i} in neurite tip. The results obtained in the present study and our previous studies together strongly suggest that high [Cl{sup −}]{sub i} in neurite tip region is crucial for efficient neurite outgrowth. - Highlights: • Intracellular Cl{sup −} concentrations ([Cl{sup −}]{sub i}) in the tip of growing neurite is higher than that in cell body in a single cell. • There is a significant positive correlation between the length of growing neurite and [Cl{sup −}]{sub i} in neurite tip. • Bumetanide significantly inhibits neurite outgrowth and decreased [Cl{sup −}]{sub i} in neurite tip. • High [Cl{sup −}]{sub i} in neurite tip region is crucial for efficient neurite outgrowth.« less

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 (H 2 O 2 ). Shh alleviated the apoptosis rate of H 2 O 2 -induced neurons. Shh also increased neuritogenesis injuried by H 2 O 2 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 H 2 O 2 . In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H 2 O 2 -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

The protection of acetylcholinesterase inhibitor on β-amyloid-induced injury of neurite outgrowth via regulating axon guidance related genes expression in neuronal cells

PubMed Central

Shen, Jiao-Ning; Wang, Deng-Shun; Wang, Rui

2012-01-01

Cognitive deficits in AD correlate with progressive synaptic dysfunction and loss. The Rho family of small GTPases, including Rho, Rac, and Cdc42, has a central role in cellular motility and cytokinesis. Acetylcholinesterase inhibitor has been found to protect cells against a broad range of reagents-induced injuries. Present studies examined if the effect of HupA on neurite outgrowth in Aβ-treated neuronal cells executed via regulating Rho-GTPase mediated axon guidance relative gene expression. Affymetrix cDNA microarray assay followed by real-time RT-PCR and Western Blotting analysis were used to elucidate and analyze the signaling pathway involved in Aβ and HupA’s effects. The effects of Aβ and HupA on the neurite outgrowth were further confirmed via immunofluorescence staining. Aβ up-regulated the mRNA expressions of NFAT5, LIMK1, EPHA1, NTN4 and RAC2 markedly in SH-SY5Y cells. Co-incubation of Aβ and HupA reversed or decreased the changes of NFAT5, NTN4, RAC2, CDC42 and SEMA4F. HupA treated alone increased NFAT5, LIMK1, NTN4 significantly. Following qRT-PCR validation showed that the correlation of the gene expression ratio between microarray and qRT-PCR is significant. Western blot result showed that the change of CDC42 protein is consistent with the mRNA level while RAC2 is not. The morphological results confirmed that HupA improved, or partly reversed, the Aβ-induced damage of neurite outgrowth. The protective effect of HupA from Aβ induced morphological injury might be correlative to, at least partially, regulating the network of neurite outgrowth related genes. PMID:23119107

Neurotrophic Effect of Citrus 5-Hydroxy-3,6,7,8,3′,4′-Hexamethoxyflavone: Promotion of Neurite Outgrowth via cAMP/PKA/CREB Pathway in PC12 Cells

PubMed Central

Lai, Hui-Chi; Wu, Ming-Jiuan; Chen, Pei-Yi; Sheu, Ting-Ting; Chiu, Szu-Ping; Lin, Meng-Han; Ho, Chi-Tang; Yen, Jui-Hung

2011-01-01

5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF), a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43). 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB) phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501), a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA) inhibitor, but not MEK1/2, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) or calcium/calmodulin-dependent protein kinase (CaMK) inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action. PMID:22140566

NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition

PubMed Central

Caltharp, Shelley A; Pira, Charmaine U; Mishima, Noboru; Youngdale, Erik N; McNeill, David S; Liwnicz, Boleslaw H; Oberg, Kerby C

2007-01-01

Background Nogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS) and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition. Results We isolated chicken NOGO-A and determined its sequence. A multiple alignment of the amino acid sequence across divergent species, identified five previously undescribed, Nogo-A specific conserved regions that may be relevant for development. NOGO gene transcripts (NOGO-A, NOGO-B and NOGO-C) were differentially expressed in the CNS during development and a second NOGO-A splice variant was identified. We further localized NOGO-A expression during key phases of CNS development by in situ hybridization. CNS-associated NOGO-A was induced coincident with neural plate formation and up-regulated by FGF in the transformation of non-neural ectoderm into neural precursors. NOGO-A expression was diffuse in the neuroectoderm during the early proliferative phase of development, and migration, but localized to large projection neurons of the optic tectum and tectal-associated nuclei during architectural differentiation, lamination and network establishment. Conclusion These data suggest Nogo-A plays a functional role in the determination of neural identity and/or differentiation and also appears to play a later role in the networking of large projection neurons during neurite formation and synaptogenesis. These data indicate that Nogo-A is a multifunctional protein with additional roles during CNS development that are disparate from its later role of neurite outgrowth inhibition in the adult CNS. PMID:17433109

A hypothetical mechanism of intraepidermal neurite formation in NC/Nga mice with atopic dermatitis.

PubMed

Tominaga, Mitsutoshi; Ozawa, Sumiko; Ogawa, Hideoki; Takamori, Kenji

2007-06-01

Pruritus is a symptom in atopic dermatitis (AD). Previous studies have reported that increased intraepidermal neurites are observed in AD, suggesting that the neuritogenesis is related to itching in the skin. This study was conducted to reveal the mechanism of intraepidermal neurite formation in AD. In this study, we used conventional (Conv) NC/Nga mice with AD. NC/Nga mice maintained in specific pathogen-free (SPF) condition were used as a control with no AD. Distribution of intraepidermal neurites and expression patterns of growth factors (NGF and amphiregulin (AR)) and cell-cell junctional molecules (E-cadherin, zona occludens 1 (ZO-1) and desmoglein 3 (Dsg3)) were examined in the skins by immunohistochemistry or quantitative RT-PCR. Furthermore, detection of gelatinase activity was performed with in situ zymography. The same experiments were conducted in ICR mice for comparison with NC/Nga mice. Neurite density and expression levels of growth factors and gelatinase were remarkably increased in the epidermis of Conv-NC/Nga mice compared with those of SPF-NC/Nga mice. Decreased expression of E-cadherin and ZO-1 and misexpression of Dsg3 were also observed in the atopic skins. In comparison with ICR mice, increases of neurite density and gelatinase activity were found in the skins of SPF-NC/Nga mice but expression levels of growth factors and cell-cell junctional molecules were unchanged. Increases of growth factors and gelatinase activity may be related to neurite outgrowth in the epidermis of atopic NC/Nga mice. Additionally, abnormal expressions of cell-cell junctional molecules in the epidermis may provide intercellular spaces for the neurite formation.

A characteristic chondroitin sulfate trisaccharide unit with a sulfated fucose branch exhibits neurite outgrowth-promoting activity: Novel biological roles of fucosylated chondroitin sulfates isolated from the sea cucumber Apostichopus japonicus.

PubMed

Shida, Miharu; Mikami, Tadahisa; Tamura, Jun-Ichi; Kitagawa, Hiroshi

2017-06-03

Chondroitin sulfate (CS) is a class of sulfated glycosaminoglycan (GAG) chains that consist of repeating disaccharide unit composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). CS chains are found throughout the pericellular and extracellular spaces and contribute to the formation of functional microenvironments for numerous biological events. However, their structure-function relations remain to be fully characterized. Here, a fucosylated CS (FCS) was isolated from the body wall of the sea cucumber Apostichopus japonicus. Its promotional effects on neurite outgrowth were assessed by using isolated polysaccharides and the chemically synthesized FCS trisaccharide β-D-GalNAc(4,6-O-disulfate) (1-4)[α-l-fucose (2,4-O-disulfate) (1-3)]-β-D-GlcA. FCS polysaccharides contained the E-type disaccharide unit GlcA-GalNAc(4,6-O-disulfate) as a CS major backbone structure and carried distinct sulfated fucose branches. Despite their relatively lower abundance of E unit, FCS polysaccharides exhibited neurite outgrowth-promoting activity comparable to squid cartilage-derived CS-E polysaccharides, which are characterized by their predominant E units, suggesting potential roles of the fucose branch in neurite outgrowth. Indeed, the chemically synthesized FCS trisaccharide was as effective as CS-E tetrasaccharide in stimulating neurite elongation in vitro. In conclusion, FCS trisaccharide units with 2,4-O-disulfated fucose branches may provide new insights into understanding the structure-function relations of CS chains. Copyright © 2017 Elsevier Inc. All rights reserved.

The protection of acetylcholinesterase inhibitor on β-amyloid-induced the injury of neurite outgrowth via regulating axon guidance related genes expression in neuronal cells.

PubMed

Shen, Jiao-Ning; Wang, Deng-Shun; Wang, Rui

2012-01-01

Cognitive deficits in AD correlate with progressive synaptic dysfunction and loss. The Rho family of small GTPases, including Rho, Rac, and Cdc42, has a central role in cellular motility and cytokinesis. Acetylcholinesterase inhibitor has been found to protect cells against a broad range of reagents-induced injuries. Present studies examined if the effect of HupA on neurite outgrowth in Aβ-treated neuronal cells executed via regulating Rho-GTPase mediated axon guidance relative gene expression. Affymetrix cDNA microarray assay followed by real-time RT-PCR and Western Blotting analysis were used to elucidate and analyze the signaling pathway involved in Aβ and HupA's effects. The effects of Aβ and HupA on the neurite outgrowth were further confirmed via immunofluorescence staining. Aβ up-regulated the mRNA expressions of NFAT5, LIMK1, EPHA1, NTN4 and RAC2 markedly in SH-SY5Y cells. Co-incubation of Aβ and HupA reversed or decreased the changes of NFAT5, NTN4, RAC2, CDC42 and SEMA4F. HupA treated alone increased NFAT5, LIMK1, NTN4 significantly. Following qRT-PCR validation showed that the correlation of the gene expression ratio between microarray and qRT-PCR is significant. Western blot result showed that the change of CDC42 protein is consistent with the mRNA level while RAC2 is not. The morphological results confirmed that HupA improved, or partly reversed, the Aβ-induced damage of neurite outgrowth. The protective effect of HupA from Aβ induced morphological injury might be correlative to, at least partially, regulating the network of neurite outgrowth related genes.

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

PubMed Central

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

2014-01-01

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

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-01-01

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

Foxp2 Regulates Gene Networks Implicated in Neurite Outgrowth in the Developing Brain

PubMed Central

Vernes, Sonja C.; Oliver, Peter L.; Spiteri, Elizabeth; Lockstone, Helen E.; Puliyadi, Rathi; Taylor, Jennifer M.; Ho, Joses; Mombereau, Cedric; Brewer, Ariel; Lowy, Ernesto; Nicod, Jérôme; Groszer, Matthias; Baban, Dilair; Sahgal, Natasha; Cazier, Jean-Baptiste; Ragoussis, Jiannis; Davies, Kay E.; Geschwind, Daniel H.; Fisher, Simon E.

2011-01-01

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections. PMID:21765815

Foxp2 regulates gene networks implicated in neurite outgrowth in the developing brain.

PubMed

Vernes, Sonja C; Oliver, Peter L; Spiteri, Elizabeth; Lockstone, Helen E; Puliyadi, Rathi; Taylor, Jennifer M; Ho, Joses; Mombereau, Cedric; Brewer, Ariel; Lowy, Ernesto; Nicod, Jérôme; Groszer, Matthias; Baban, Dilair; Sahgal, Natasha; Cazier, Jean-Baptiste; Ragoussis, Jiannis; Davies, Kay E; Geschwind, Daniel H; Fisher, Simon E

2011-07-01

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP-chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujimura, Masatake, E-mail: fujimura@nimd.go.jp; Usuki, Fusako; Cheng, Jinping

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

Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons

PubMed Central

Vernon, Claire G.

2017-01-01

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG–dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2−/− neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth. SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG–dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Dong-Hee; Department of Medical Science, Konkuk University School of Medicine, Seoul; Lee, Kyoung-Hee

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, wemore » 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

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

PubMed

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

2014-02-27

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

Neurite differentiation is modulated in neuroblastoma cells engineered for altered acetylcholinesterase expression.

PubMed

Koenigsberger, C; Chiappa, S; Brimijoin, S

1997-10-01

Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level (p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.

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

Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons.

PubMed

Vernon, Claire G; Swanson, Geoffrey T

2017-03-22

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2 -/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth. SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and

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

E-cadherin can replace N-cadherin during secretory-stage enamel development.

PubMed

Guan, Xiaomu; Bidlack, Felicitas B; Stokes, Nicole; Bartlett, John D

2014-01-01

N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development. The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. μCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ. The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous

Suppression of neurite outgrowth of primary cultured hippocampal neurons is involved in impairment of glutamate metabolism and NMDA receptor function caused by nanoparticulate TiO2.

PubMed

Hong, Fashui; Sheng, Lei; Ze, Yuguan; Hong, Jie; Zhou, Yingjun; Wang, Ling; Liu, Dong; Yu, Xiaohong; Xu, Bingqing; Zhao, Xiaoyang; Ze, Xiao

2015-06-01

Numerous studies have indicated that nano-titanium dioxide (TiO2) can induce neurotoxicity in vitro and in vivo, however, it is unclear whether nano-TiO2 affects neurite outgrowth of hippocampal neurons. In order to investigate the mechanism of neurotoxicity, rat primary cultured hippocampal neurons on the fourth day of culture were exposed to 5, 15, and 30 μg/mL nano-TiO2 for 24 h, and nano-TiO2 internalization, dendritic growth, glutamate metabolism, expression of N-methyl-D-aspartate (NMDA) receptor subunits (NR1, NR2A and NR2B), calcium homeostasis, sodium current (INa) and potassium current (IK) were examined. Our findings demonstrated that nano-TiO2 crossed the membrane into the cytoplasm or nucleus, and significantly suppressed dendritic growth of primary cultured hippocampal neurons in a concentration-dependent manner. Furthermore, nano-TiO2 induced a marked release of glutamate to the extracellular region, decreased glutamine synthetase activity and increased phosphate-activated glutaminase activity, elevated intracellular calcium ([Ca(2+)]i), down-regulated protein expression of NR1, NR2A and NR2B, and increased the amplitudes of the INa and IK. In addition, nano-TiO2 increased nitric oxide and nitrice synthase, attenuated the activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase, and increased the ADP/ATP ratio in the primary neurons. Taken together, these findings indicate that nano-TiO2 inhibits neurite outgrowth of hippocampal neurons by interfering with glutamate metabolism and impairing NMDA receptor function. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sub-toxic concentrations of nano-ZnO and nano-TiO2 suppress neurite outgrowth in differentiated PC12 cells.

PubMed

Irie, Tomohiko; Kawakami, Tsuyoshi; Sato, Kaoru; Usami, Makoto

2017-01-01

Nanomaterials have been extensively used in our daily life, and may also induce health effects and toxicity. Nanomaterials can translocate from the outside to internal organs, including the brain. For example, both nano-ZnO and nano-TiO 2 translocate into the brain via the olfactory pathway in rodents, possibly leading to toxic effects on the brain. Although the effects of nano-ZnO and nano-TiO 2 on neuronal viability or neuronal excitability have been studied, no work has focused on how these nanomaterials affect neuronal differentiation and development. In this study, we investigated the effects of nano-ZnO and nano-TiO 2 on neurite outgrowth of PC12 cells, a useful model system for neuronal differentiation. Surprisingly, the number, length, and branching of differentiated PC12 neurites were significantly suppressed by the 7-day exposure to nano-ZnO (in the range of 1.0 × 10 -4 to 1.0 × 10 -1 µg/mL), at which the cell viability was not affected. The number and length were also significantly inhibited by the 7-day exposure to nano-TiO 2 (1.0 × 10 -3 to 1.0 µg/mL), which did not have cytotoxic effects. These results demonstrate that the neurite outgrowth in differentiated PC12 cells was suppressed by sub-cytotoxic concentrations of nano-ZnO or nano-TiO 2 .

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

PubMed Central

Yu, Minrui; Huang, Yu; Ballweg, Jason; Shin, Hyuncheol; Huang, Minghuang; Savage, Donald E.; Lagally, Max G.; Dent, Erik W.; Blick, Robert H.; Williams, Justin C.

2013-01-01

In many neural culture studies, neurite migration on a flat, open surface does not reflect the three-dimensional (3D) microenvironment in vivo. With that in mind, we fabricated arrays of semiconductor tubes using strained silicon (Si) and germanium (Ge) nanomembranes and employed them as a cell culture substrate for primary cortical neurons. Our experiments show that the SiGe substrate and the tube fabrication process are biologically viable for neuron cells. We also observe that neurons are attracted by the tube topography, even in the absence of adhesion factors, and can be guided to pass through the tubes during outgrowth. Coupled with selective seeding of individual neurons close to the tube opening, growth within a tube can be limited to a single axon. Furthermore, the tube feature resembles the natural myelin, both physically and electrically, and it is possible to control the tube diameter to be close to that of an axon, providing a confined 3D contact with the axon membrane and potentially insulating it from the extracellular solution. PMID:21366271

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

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

PubMed

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

2016-05-21

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

Assembly and turnover of neurofilaments in growing axonal neurites.

PubMed

Boumil, Edward F; Vohnoutka, Rishel; Lee, Sangmook; Pant, Harish; Shea, Thomas B

2018-01-26

Neurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites ('bundled NFs'). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered. © 2018. Published by The Company of Biologists Ltd.

Assembly and turnover of neurofilaments in growing axonal neurites

PubMed Central

Boumil, Edward F.; Vohnoutka, Rishel; Lee, Sangmook; Pant, Harish

2018-01-01

ABSTRACT Neurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites (‘bundled NFs’). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered. PMID:29158321

Differential regulation of axon outgrowth and reinnervation by neurotrophin-3 and neurotrophin-4 in the hippocampal formation.

PubMed

Hechler, Daniel; Boato, Francesco; Nitsch, Robert; Hendrix, Sven

2010-08-01

In this study, we investigated the hypothesis whether neurotrophins have a differential influence on neurite growth from the entorhinal cortex depending on the presence or absence of hippocampal target tissue. We investigated organotypic brain slices derived from the entorhinal-hippocampal system to analyze the effects of endogenous and recombinant neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) on neurite outgrowth and reinnervation. In the reinnervation assay, entorhinal cortex explants of transgenic mice expressing enhanced green fluorescent protein (EGFP) were co-cultured with wild-type hippocampi under the influence of recombinant NT-3 and NT-4 (500 ng/ml). Both recombinant NT-3 and NT-4 significantly increased the growth of EGFP+ nerve fibers into the target tissue. Consistently, reinnervation of the hippocampi of NT-4(-/-) and NT-3(+/-)NT-4(-/-) mice was substantially reduced. In contrast, the outgrowth assay did not exhibit reduction in axon outgrowth of NT-4(-/-) or NT-3(+/-)NT-4(-/-) cortex explants, while the application of recombinant NT-3 (500 ng/ml) induced a significant increase in the neurite extension of cortex explants. Recombinant NT-4 had no effect. In summary, only recombinant NT-3 stimulates axon outgrowth from cortex explants, while both endogenous and recombinant NT-3 and NT-4 synergistically promote reinnervation of the denervated hippocampus. These results suggest that endogenous and exogenous NT-3 and NT-4 differentially influence neurite growth depending on the presence or absence of target tissue.

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.

The Adiponectin Homolog Osmotin Enhances Neurite Outgrowth and Synaptic Complexity via AdipoR1/NgR1 Signaling in Alzheimer's Disease.

PubMed

Yoon, Gwangho; Shah, Shahid Ali; Ali, Tahir; Kim, Myeong Ok

2018-01-15

Alzheimer's disease is a major neurodegenerative disease characterized by memory loss and cognitive deficits. Recently, we reported that osmotin, which is a homolog of adiponectin, improved long-term potentiation and cognitive functions in Alzheimer's disease mice. Several lines of evidence have suggested that Nogo-A and the Nogo-66 receptor 1 (NgR1), which form a complex that inhibits long-term potentiation and cognitive function, might be associated with the adiponectin receptor 1 (AdipoR1), which is a receptor for osmotin. Here, we explore whether osmotin's effects on long-term potentiation and memory function are associated with NgR1 signaling via AdipoR1 in Alzheimer's disease. Osmotin reduced the expression of NgR1 without affecting Nogo-A expression. Furthermore, osmotin inhibited NgR1 signaling by prohibiting the formation of the Nogo-A and NgR1 ligand-receptor complex, resulting in enhanced neurite outgrowth; these effects disappeared in the presence of AdipoR1 interference. In addition, osmotin increased the expression of the pre- and postsynaptic markers synaptophysin and PSD-95, as well as the activation of the memory-associated markers AMPA receptor and CREB; these effects occurred in an AdipoR1- and NgR1-dependent manner. Osmotin was also found to enhance dendritic complexity and spine density in the hippocampal region of Alzheimer's disease mouse brains. These results suggest that osmotin can enhance neurite outgrowth and synaptic complexity through AdipoR1 and NgR1 signaling, implying that osmotin might be an effective therapeutic agent for Alzheimer's disease and that AdipoR1 might be a crucial therapeutic target for neurodegenerative diseases such as Alzheimer's.

Differential Function of N-Cadherin and Cadherin-7 in the Control of Embryonic Cell Motility

PubMed Central

Dufour, Sylvie; Beauvais-Jouneau, Alice; Delouvée, Annie; Thiery, Jean Paul

1999-01-01

Similar amounts of N-cadherin and cadherin-7, the prototypes of type I and type II cadherin, induced cell-cell adhesion in murine sarcoma 180 transfectants, Ncad-1 and cad7-29, respectively. However, in the initial phase of aggregation, Ncad-1 cells aggregated more rapidly than cad7-29 cells. Isolated Ncad-1 and cad7-29 cells adhered and spread in a similar manner on fibronectin (FN), whereas aggregated cad7-29 cells were more motile and dispersed than aggregated Ncad-1 cells. cad7-29 cells established transient contacts with their neighbors which were stabilized if FN-cell interactions were perturbed. In contrast, Ncad-1 cells remained in close contact when they migrated on FN. Both β-catenin and cadherin were more rapidly downregulated in cad7-29 than in Ncad-1 cells treated with cycloheximide, suggesting a higher turnover rate for cadherin-7–mediated cell-cell contacts than for those mediated by N-cadherin. The extent of FN-dependent focal adhesion kinase phosphorylation was much lower if the cells had initiated N-cadherin–mediated rather than cadherin-7–mediated cell adhesion before plating. On grafting into the embryo, Ncad-1 cells did not migrate and remained at or close to the graft site, even after 48 h, whereas grafted cad7-29 cells dispersed efficiently into embryonic structures. Thus, the adhesive phenotype of cadherin-7–expressing cells is regulated by the nature of the extracellular matrix environment which also controls the migratory behavior of the cells. In addition, adhesions mediated by different cadherins differentially regulate FN-dependent signaling. The transient contacts specifically observed in cadherin- 7–expressing cells may also be important in the control of cell motility. PMID:10427101

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

PubMed

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

2017-10-01

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

Complement Protein C1q Modulates Neurite Outgrowth In Vitro and Spinal Cord Axon Regeneration In Vivo

PubMed Central

Peterson, Sheri L.; Nguyen, Hal X.; Mendez, Oscar A.

2015-01-01

Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI. PMID:25762679

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

PubMed

Xing, Dongming; Ma, Lie; Gao, Changyou

2014-10-01

The modification of biodegradable polyesters with bioactive molecules has become an important strategy for controlling neuron adhesion and neurite outgrowth in nerve regeneration. In this study we report a biodegradable poly(ester-carbonate) with a pendant acetylcholine analog, which a neurotransmitter for the enhancement of neuron adhesion and outgrowth. The acetylcholine-functionalized poly(ester-carbonate) (Ach-P(LA-ClTMC)) was prepared by copolymerizing l-lactide (LA) and 5-methyl-5-chloroethoxycarbonyl trimethylene carbonate (ClTMC), followed by quaternization with trimethylamine. The acetylcholine analog content could be modulated by changing the molar feeding fraction of ClTMC. The incorporation of the acetylcholine analog improved the hydrophilicity of the films, but the acetylcholine analog content did not significantly influence the surface morphology of the acetylcholine-functionalized films. The results of PC12 cell culture showed that the acetylcholine analog promoted cell viability and neurite outgrowth in a concentration-dependent manner. The longest length of neurite and the percentage of cells bearing neurites were obtained on the Ach-P(LA-ClTMC)-10 film. All the results indicate that the integration of the acetylcholine analog at an appropriate fraction could be an effective strategy for optimizing the existing biodegradable polyesters for nerve regeneration applications. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

E-cadherin and, in its absence, N-cadherin promotes Nanog expression in mouse embryonic stem cells via STAT3 phosphorylation.

PubMed

Hawkins, Kate; Mohamet, Lisa; Ritson, Sarah; Merry, Catherine L R; Ward, Christopher M

2012-09-01

We have recently shown that loss of E-cadherin in mouse embryonic stem cells (mESCs) results in significant alterations to both the transcriptome and hierarchy of pluripotency-associated signaling pathways. Here, we show that E-cadherin promotes kruppel-like factor 4 (Klf4) and Nanog transcript and protein expression in mESCs via STAT3 phosphorylation and that β-catenin, and its binding region in E-cadherin, is required for this function. To further investigate the role of E-cadherin in leukemia inhibitory factor (LIF)-dependent pluripotency, E-cadherin null (Ecad(-/-)) mESCs were cultured in LIF/bone morphogenetic protein supplemented medium. Under these conditions, Ecad(-/-) mESCs exhibited partial restoration of cell-cell contact and STAT3 phosphorylation and upregulated Klf4, Nanog, and N-cadherin transcripts and protein. Abrogation of N-cadherin using an inhibitory peptide caused loss of phospho STAT3, Klf4, and Nanog in these cells, demonstrating that N-cadherin supports LIF-dependent pluripotency in this context. We therefore identify a novel molecular mechanism linking E- and N-cadherin to the core circuitry of pluripotency in mESCs. This mechanism may explain the recently documented role of E-cadherin in efficient induced pluripotent stem cell reprogramming. Copyright © 2012 AlphaMed Press.

Robust Neurite Extension Following Exogenous Electrical Stimulation within Single Walled Carbon Nanotube-Composite Hydrogels

PubMed Central

Koppes, A. N.; Keating, K. W.; McGregor, A. L.; Koppes, R. A.; Kearns, K. R.; Ziemba, A. M.; McKay, C. A.; Zuidema, J. M.; Rivet, C. J.; Gilbert, R. J.; Thompson, D. M.

2016-01-01

The use of exogenous electrical stimulation to promote nerve regeneration has achieved only limited success. Conditions impeding optimized outgrowth may arise from inadequate stimulus presentation due to differences in injury geometry or signal attenuation. Implantation of an electrically-conductive biomaterial may mitigate this attenuation and provide a more reproducible signal. In this study, a conductive nanofiller (single-walled carbon nanotubes [SWCNT]) was selected as one possible material to manipulate the bulk electrical properties of a collagen type I-10% Matrigel™ composite hydrogel. Neurite outgrowth within hydrogels (SWCNT or nanofiller-free controls) was characterized to determine if: 1) nanofillers influence neurite extension and 2) electrical stimulation of the nanofiller composite hydrogel enhances neurite outgrowth. Increased SWCNT loading (10–100-μg/ml) resulted in greater bulk conductivity (up to 1.7-fold) with no significant changes to elastic modulus. Neurite outgrowth increased 3.3-fold in 20-μg/mL SWCNT loaded biomaterials relative to the nanofiller-free control. Electrical stimulation promoted greater outgrowth (2.9-fold) within SWCNT-free control. The concurrent presentation of electrical stimulation and SWCNT-loaded biomaterials resulted in a 7.0-fold increase in outgrowth relative to the unstimulated, nanofiller-free controls. Local glia residing within the DRG likely contribute, in part, to the observed increases in outgrowth; but it is unknown which specific nanofiller properties influence neurite extension. Characterization of neuronal behavior in model systems, such as those described here, will aid the rational development of biomaterials as well as the appropriate delivery of electrical stimuli to support nerve repair. PMID:27167609

DDX3 binding with CK1ε was closely related to motor neuron degeneration of ALS by affecting neurite outgrowth.

PubMed

Chen, Yanchun; Wang, Qing; Wang, Qiaozhen; Liu, Huancai; Zhou, Fenghua; Zhang, Yawen; Yuan, Meng; Zhao, Chunyan; Guan, Yingjun; Wang, Xin

2017-01-01

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease characterized by progressive degeneration of motor neurons. The pathogenesis of ALS remains largely unknown. RNA helicase DDX3 is a multifunctional protein involved in several steps of gene expression. Casein kinase 1ε (CK1ε) is an important signal molecule of Wnt signaling pathway and is closely related to neurite growth. However, the roles of DDX3 and CK1ε in the pathogenesis of ALS remain unclear. In this study, we first investigated the expression of DDX3 and CK1ε in the spinal cord of SOD1-G93A ALS transgenic mice using RT-PCR, Western blot and immunohistochemical technique. Results showed that the altered expression of DDX3 and CK1ε was found in the spinal cord of ALS mice. DDX3 and CK1ε positive cells were mainly distributed in the anterior horn of spinal cord and co-localized with neurons not with glial cells, suggesting that the altered expression of DDX3 and CK1ε was closely related to motor neuron degeneration of ALS. Moreover, we selected NSC34 cell line and transfected pEGFP-G93A-SOD1 plasmid to further examine the mechanism. Knockdown of DDX3 that uses small interfering RNA (siRNA) decreased the mRNA and protein levels of CK1ε significantly and inhibited neurite outgrowth of SOD1 mutant NSC34 cells in vitro. Co-immunoprecipitation kit confirmed that DDX3 could band with CK1ε in vivo. Our data suggested that DDX3 binding with CK1ε was closely related to motor neuron degeneration of ALS by affecting neurite outgrowth. Thus, elucidating the underlying mechanisms of ALS is crucial for future development of ALS treatments.

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.

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.

Critical time window of neuronal cholesterol synthesis during neurite outgrowth.

PubMed

Fünfschilling, Ursula; Jockusch, Wolf J; Sivakumar, Nandhini; Möbius, Wiebke; Corthals, Kristina; Li, Sai; Quintes, Susanne; Kim, Younghoon; Schaap, Iwan A T; Rhee, Jeong-Seop; Nave, Klaus-Armin; Saher, Gesine

2012-05-30

Cholesterol is an essential membrane component enriched in plasma membranes, growth cones, and synapses. The brain normally synthesizes all cholesterol locally, but the contribution of individual cell types to brain cholesterol metabolism is unknown. To investigate whether cortical projection neurons in vivo essentially require cholesterol biosynthesis and which cell types support neurons, we have conditionally ablated the cholesterol biosynthesis in these neurons in mice either embryonically or postnatally. We found that cortical projection neurons synthesize cholesterol during their entire lifetime. At all stages, they can also benefit from glial support. Adult neurons that lack cholesterol biosynthesis are mainly supported by astrocytes such that their functional integrity is preserved. In contrast, microglial cells support young neurons. However, compensatory efforts of microglia are only transient leading to layer-specific neuronal death and the reduction of cortical projections. Hence, during the phase of maximal membrane growth and maximal cholesterol demand, neuronal cholesterol biosynthesis is indispensable. Analysis of primary neurons revealed that neurons tolerate only slight alteration in the cholesterol content and plasma membrane tension. This quality control allows neurons to differentiate normally and adjusts the extent of neurite outgrowth, the number of functional growth cones and synapses to the available cholesterol. This study highlights both the flexibility and the limits of horizontal cholesterol transfer in vivo and may have implications for the understanding of neurodegenerative diseases.

Guidance of neurite outgrowth on aligned electrospun polypyrrole/poly(styrene-beta-isobutylene-beta-styrene) fiber platforms.

PubMed

Liu, Xiao; Chen, Jun; Gilmore, Kerry J; Higgins, Michael J; Liu, Yong; Wallace, Gordon G

2010-09-15

The purpose of this work was to investigate the potential biomedical application of novel aligned electrospun polypyrrole (PPy)/poly(styrene-beta-isobutylene-beta-styrene) (SIBS) fibers. After successfully aligning the electroactive PPy/SIBS fibers based on our modified electrospinning method, we demonstrated that neurite outgrowth from PC12 cells could be highly orientated parallel to the aligned PPy/SIBS fibers. Physical interactions between the nerve cells and PPy/SIBS fibers through filopodia "sensing" were observed using atomic force microscopy. These observations indicate a role of contact guidance as a mechanism for the observed alignment. This work highlights the capacity for electroactive PPy/SIBS fibers to support and guide nerve cell differentiation through topographic cues, which is a highly desirable characteristic in medical implants for neurological applications. (c) 2010 Wiley Periodicals, Inc.

Transcallosal Projections Require Glycoprotein M6-Dependent Neurite Growth and Guidance.

PubMed

Mita, Sakura; de Monasterio-Schrader, Patricia; Fünfschilling, Ursula; Kawasaki, Takahiko; Mizuno, Hidenobu; Iwasato, Takuji; Nave, Klaus-Armin; Werner, Hauke B; Hirata, Tatsumi

2015-11-01

The function of mature neurons critically relies on the developmental outgrowth and projection of their cellular processes. It has long been postulated that the neuronal glycoproteins M6a and M6b are involved in axon growth because these four-transmembrane domain-proteins of the proteolipid protein family are highly enriched on growth cones, but in vivo evidence has been lacking. Here, we report that the function of M6 proteins is required for normal axonal extension and guidance in vivo. In mice lacking both M6a and M6b, a severe hypoplasia of axon tracts was manifested. Most strikingly, the corpus callosum was reduced in thickness despite normal densities of cortical projection neurons. In single neuron tracing, many axons appeared shorter and disorganized in the double-mutant cortex, and some of them were even misdirected laterally toward the subcortex. Probst bundles were not observed. Upon culturing, double-mutant cortical and cerebellar neurons displayed impaired neurite outgrowth, indicating a cell-intrinsic function of M6 proteins. A rescue experiment showed that the intracellular loop of M6a is essential for the support of neurite extension. We propose that M6 proteins are required for proper extension and guidance of callosal axons that follow one of the most complex trajectories in the mammalian nervous system. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Ectodomain shedding of Limbic System-Associated Membrane Protein (LSAMP) by ADAM Metallopeptidases promotes neurite outgrowth in DRG neurons.

PubMed

Sanz, Ricardo L; Ferraro, Gino B; Girouard, Marie-Pier; Fournier, Alyson E

2017-08-11

IgLONs are members of the immunoglobulin superfamily of cell adhesion proteins implicated in the process of neuronal outgrowth, cell adhesion and subdomain target recognition. IgLONs form homophilic and heterophilic complexes on the cell surface that repress or promote growth depending on the neuronal population, the developmental stage and surface repertoire of IgLON family members. In the present study, we identified a metalloproteinase-dependent mechanism necessary to promote growth in embryonic dorsal root ganglion cells (DRGs). Treatment of embryonic DRG neurons with pan-metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-3, or an inhibitor of ADAM Metallopeptidase Domain 10 (ADAM10) reduces outgrowth from DRG neurons indicating that metalloproteinase activity is important for outgrowth. The IgLON family members Neurotrimin (NTM) and Limbic System-Associated Membrane Protein (LSAMP) were identified as ADAM10 substrates that are shed from the cell surface of DRG neurons. Overexpression of LSAMP and NTM suppresses outgrowth from DRG neurons. Furthermore, LSAMP loss of function decreases the outgrowth sensitivity to an ADAM10 inhibitor. Together our findings support a role for ADAM-dependent shedding of cell surface LSAMP in promoting outgrowth from DRG neurons.

N-cadherin is required for cytodifferentiation during zebrafish odontogenesis.

PubMed

Verstraeten, B; van Hengel, J; Sanders, E; Van Roy, F; Huysseune, A

2013-04-01

N-cadherin is a well-studied classic cadherin involved in multiple developmental processes and is also known to have a signaling function. Using the zebrafish (Danio rerio) as a model, we tested the hypothesis that tooth morphogenesis is accompanied by dynamic changes in N-cadherin distribution and that absence of N-cadherin disturbs tooth development. N-cadherin, encoded by the gene cdh2, is absent during the initiation and morphogenesis stages of both primary (first-generation) and replacement teeth, as demonstrated by immunohistochemistry. However, N-cadherin is up-regulated at the onset of differentiation of cells of the inner dental epithelium and the dental papilla, i.e., the ameloblasts and odontoblasts, respectively. In the inner dental epithelium, N-cadherin is co-expressed with E-cadherin, excluding the occurrence of cadherin switching such as observed during human tooth development. While early lethality of N-cadherin knockout mice prevents any functional study of N-cadherin in mouse odontogenesis, zebrafish parachute (pac) mutants, deficient for N-cadherin, survive beyond the age when primary teeth normally start to form. In these mutants, the first tooth forms, but its development stops at the early cytodifferentiation stage. N-cadherin deficiency also completely inhibits the development of the other first-generation teeth, possibly due to the absence of N-cadherin signaling once the first tooth has differentiated.

Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

PubMed Central

Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide

2013-01-01

Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca2+ signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB. PMID:24116072

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.

Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities.

PubMed

Nagase, Hiroyuki; Omae, Naoki; Omori, Akiko; Nakagawasai, Osamu; Tadano, Takeshi; Yokosuka, Akihito; Sashida, Yutaka; Mimaki, Yoshihiro; Yamakuni, Tohru; Ohizumi, Yasushi

2005-12-02

cAMP response element (CRE) transcription is dysregulated in neurodegenerative disorders in the central nervous system (CNS), including polyglutamine diseases. As the first step to find natural compounds with protective action against neurodegeneration in the CNS, we here examined whether six citrus flavonoids, namely nobiletin, 5-demethylnobiletin, tangeretin, sinensetin, 6-demethoxytangeretin, and 6-demethoxynobiletin, stimulated CRE-dependent transcription and induced neurite outgrowth in PC12D cells. Among the compounds, nobiletin most potently enhanced CRE-dependent transcription and neurite outgrowth by activating ERK/MAP kinase-dependent signalling to increase CREB phosphorylation. The transcription and neurite outgrowth were stimulated by nobiletin in a concentration-dependent manner, with a strong correlation between them. Furthermore, a 11-day oral administration of nobiletin rescued impaired memory in olfactory-bulbectomized mice documented to be accompanied by a cholinergic neurodegeneration. These results suggest that nobiletin with the activity to improve impaired memory may become a potential leading compound for drug development for neurodegenerative disorders exhibiting the dysregulated CRE-dependent transcription.

N-cadherin Regulation of Bone Growth and Homeostasis is Osteolineage Stage-Specific

PubMed Central

Fontana, Francesca; Hickman-Brecks, Cynthia L.; Salazar, Valerie S.; Revollo, Leila; Abou-Ezzi, Grazia; Grimston, Susan K.; Jeong, Sung Yeop; Watkins, Marcus; Fortunato, Manuela; Alippe, Yael; Link, Daniel C.; Mbalaviele, Gabriel; Civitelli, Roberto

2017-01-01

N-cadherin inhibits osteogenic cell differentiation and canonical Wnt/β-catenin signaling in vitro. However, in vivo both conditional Cdh2 ablation and overexpression in osteoblasts lead to low bone mass. We tested the hypothesis that N-cadherin has different effects on osteolineage cells depending upon their differentiation stage. Embryonic conditional osteolineage Cdh2 deletion in mice results in defective growth, low bone mass and reduced osteoprogenitor number. These abnormalities are prevented by delaying Cdh2 ablation until 1 month of age, thus targeting only committed and mature osteoblasts, suggesting they are the consequence of N-cadherin deficiency in osteoprogenitors. Indeed, diaphyseal trabecularization actually increases when Cdh2 is ablated postnatally. The sclerostin-insensitive Lrp5A214V mutant, associated with high bone mass, does not rescue the growth defect, but it overrides the low bone mass of embryonically Cdh2 deleted mice, suggesting N-cadherin interacts with Wnt signaling to control bone mass. Finally, bone accrual and β-catenin accumulation after administration of an anti-Dkk1 antibody are enhanced in N-cadherin deficient mice. Thus, while lack of N-cadherin in embryonic and perinatal age is detrimental to bone growth and bone accrual, in adult mice loss of N-cadherin in osteolineage cells favors bone formation. Hence, N-cadherin inhibition may widen the therapeutic window of osteoanabolic agents. PMID:28240364

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

Regulated binding of PTP1B-like phosphatase to N-cadherin: control of cadherin-mediated adhesion by dephosphorylation of beta-catenin

PubMed Central

1996-01-01

Cadherins are a family of cell-cell adhesion molecules which play a central role in controlling morphogenetic movements during development. Cadherin function is regulated by its association with the actin containing cytoskeleton, an association mediated by a complex of cytoplasmic proteins, the catenins: alpha, beta, and gamma. Phosphorylated tyrosine residues on beta-catenin are correlated with loss of cadherin function. Consistent with this, we find that only nontyrosine phosphorylated beta-catenin is associated with N-cadherin in E10 chick retina tissue. Moreover, we demonstrate that a PTP1B-like tyrosine phosphatase associates with N-cadherin and may function as a regulatory switch controlling cadherin function by dephosphorylating beta-catenin, thereby maintaining cells in an adhesion-competent state. The PTP1B-like phosphatase is itself tyrosine phosphorylated. Moreover, both direct binding experiments performed with phosphorylated and dephosphorylated molecules, and treatment of cells with tyrosine kinase inhibitors indicate that the interaction of the PTP1B-like phosphatase with N-cadherin depends on its tyrosine phosphorylation. Concomitant with the tyrosine kinase inhibitor-induced loss of the PTP1B-like phosphatase from its association with N-cadherin, phosphorylated tyrosine residues are retained on beta-catenin, the association of N- cadherin with the actin containing cytoskeleton is lost and N-cadherin- mediated cell adhesion is prevented. Tyrosine phosphatase inhibitors also result in the accumulation of phosphorylated tyrosine residues on beta-catenin, loss of the association of N-cadherin with the actin- containing cytoskeleton, and prevent N-cadherin mediated adhesion, presumably by directly blocking the function of the PTP1B-like phosphatase. We previously showed that the binding of two ligands to the cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase), the monoclonal antibody 1B11 and a proteoglycan with a 250-kD core protein

Small-molecule inhibitors at the PSD-95/nNOS interface protect against glutamate-induced neuronal atrophy in primary cortical neurons.

PubMed

Doucet, M V; O'Toole, E; Connor, T; Harkin, A

2015-08-20

Glutamate and nitric oxide (NO) are important regulators of dendrite and axon development in the central nervous system. Excess glutamatergic stimulation is a feature of many pathological conditions and manifests in neuronal atrophy and shrinkage with eventual neurodegeneration and cell death. Here we demonstrate that treatment of cultured primary cortical rat neurons for 24h with glutamate (500μM) or N-methyl-d-aspartate (NMDA) (100-500μM) combined with glycine suppresses neurite outgrowth. A similar reduction of neurite outgrowth was observed with the NO precursor l-arginine and NO donor sodium nitroprusside (SNP) (100 and 300μM). The NMDA-receptor (NMDA-R) antagonists ketamine and MK-801 (10nM) counteracted the NMDA/glycine-induced reduction in neurite outgrowth and the neuronal NO synthase (nNOS) inhibitor 1-[2-(trifluoromethyl)phenyl] imidazole (TRIM) (100nM) counteracted both the NMDA/glycine and l-arginine-induced decreases in neurite outgrowth. Furthermore, targeting soluble guanylate cyclase (sGC), a downstream target of NO, with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10μM) also protected against l-arginine-induced decreases in neurite outgrowth. Since the NMDA-R is functionally coupled to nNOS via the postsynaptic protein 95kDa (PSD-95), inhibitors of the PSD-95/nNOS interaction were tested for their ability to protect against glutamate-induced suppression in neurite outgrowth. Treatment with the small-molecule inhibitors of the PSD-95/nNOS interface 2-((1H-benzo[d] [1,2,3]triazol-5-ylamino) methyl)-4,6-dichlorophenol (IC87201) (10 and 100nM) and 4-(3,5-dichloro-2-hydroxy-benzylamino)-2-hydroxybenzoic acid (ZL-006) (10 and 100nM) attenuated NMDA/glycine-induced decreases in neurite outgrowth. These data support the hypothesis that targeting the NMDA-R/PSD-95/nNOS interaction downstream of NMDA-R promotes neurotrophic effects by preventing neurite shrinkage in response to excess glutamatergic stimulation. The PSD-95/n

Growth Cone MKK7 mRNA Targeting Regulates MAP1b-Dependent Microtubule Bundling to Control Neurite Elongation

PubMed Central

Feltrin, Daniel; Fusco, Ludovico; Witte, Harald; Moretti, Francesca; Martin, Katrin; Letzelter, Michel; Fluri, Erika; Scheiffele, Peter; Pertz, Olivier

2012-01-01

Local mRNA translation in neurons has been mostly studied during axon guidance and synapse formation but not during initial neurite outgrowth. We performed a genome-wide screen for neurite-enriched mRNAs and identified an mRNA that encodes mitogen-activated protein kinase kinase 7 (MKK7), a MAP kinase kinase (MAPKK) for Jun kinase (JNK). We show that MKK7 mRNA localizes to the growth cone where it has the potential to be translated. MKK7 is then specifically phosphorylated in the neurite shaft, where it is part of a MAP kinase signaling module consisting of dual leucine zipper kinase (DLK), MKK7, and JNK1. This triggers Map1b phosphorylation to regulate microtubule bundling leading to neurite elongation. We propose a model in which MKK7 mRNA localization and translation in the growth cone allows for a mechanism to position JNK signaling in the neurite shaft and to specifically link it to regulation of microtubule bundling. At the same time, this uncouples activated JNK from its functions relevant to nuclear translocation and transcriptional activation. PMID:23226105

Nanostructured Polyaniline Coating on ITO Glass Promotes the Neurite Outgrowth of PC 12 Cells by Electrical Stimulation.

PubMed

Wang, Liping; Huang, Qianwei; Wang, Jin-Ye

2015-11-10

A conducting polymer polyaniline (PANI) with nanostructure was synthesized on indium tin oxide (ITO) glass. The effect of electrical stimulation on the proliferation and the length of neurites of PC 12 cells was investigated. The dynamic protein adsorption on PANI and ITO surfaces in a cell culture medium was also compared with and without electrical stimulation. The adsorbed proteins were characterized using SDS-PAGE. A PANI coating on ITO surface was shown with 30-50 nm spherical nanostructure. The number of PC 12 cells was significantly greater on the PANI/ITO surface than on ITO and plate surfaces after cell seeding for 24 and 36 h. This result confirmed that the PANI coating is nontoxic to PC 12 cells. The electrical stimulation for 1, 2, and 4 h significantly enhanced the cell numbers for both PANI and ITO conducting surfaces. Moreover, the application of electrical stimulation also improved the neurite outgrowth of PC 12 cells, and the number of PC 12 cells with longer neurite lengths increased obviously under electrical stimulation for the PANI surface. From the mechanism, the adsorption of DMEM proteins was found to be enhanced by electrical stimulation for both PANI/ITO and ITO surfaces. A new band 2 (around 37 kDa) was observed from the collected adsorbed proteins when PC 12 cells were cultured on these surfaces, and culturing PC 12 cells also seemed to increase the amount of band 1 (around 90 kDa). When immersing PANI/ITO and ITO surfaces in a DMEM medium without a cell culture, the number of band 3 (around 70 kDa) and band 4 (around 45 kDa) proteins decreased compared to that of PC 12 cell cultured surfaces. These results are valuable for the design and improvement of the material performance for neural regeneration.

A family of glycoproteins (GP55), which inhibit neurite outgrowth, are members of the Ig superfamily and are related to OBCAM, neurotrimin, LAMP and CEPU-1.

PubMed

Wilson, D J; Kim, D S; Clarke, G A; Marshall-Clarke, S; Moss, D J

1996-12-01

We have previously identified a glycosylphosphatidylinositol-linked glycoprotein of 55 kDa (GP55) which inhibits neurite outgrowth. We now provide evidence that GP55, isolated from adult chick brain, consists of at least two bands, both of which are active, i.e., block outgrowth of neurites from chick dorsal root ganglion neurons. An antiserum raised against the adult proteins reverses the inhibition and preliminary experiments suggest that GP55 is restricted to the nervous system, increases during development from very low levels at embryonic day 10 and is most abundant after hatching. Immunofluorescence reveals that GP55 is expressed on neurons cultured from an embryonic day 14 chick brain but is barely detectable on embryonic day 10 dorsal root ganglion neurons or embryonic day 8 forebrain neurons; the neurons which respond to substrate-bound GP55. Peptide sequencing revealed considerable homology with OBCAM, a protein previously identified on the basis of binding opiates. Nested polymerase chain reaction using primers to the OBCAM sequence and internal primers to GP55 peptides produced two different polymerase chain reaction fragments with homology to OBCAM. A full length clone (E19S) corresponding to one polymerase chain reaction product and a partial length clone (E14S) corresponding to the second have been isolated from an embryonic chick brain library. Both are members of the immunoglobulin superfamily and have (or are expected to have) three C2 domains. E19S has 90% homology with LAMP at the amino acid level. This sequence only partially matches the peptides from the adult protein and hence is probably not a major component of the adult proteins. E14S (GP55-A) has 83% homology to OBCAM at the amino acid level over the region sequenced. The sequence matches several of the peptides from the adult protein and is hence likely to correspond to a major component of the adult proteins. Thus members of the GP55 family are related to OBCAM, neurotrimin, LAMP and a

RhoA Inhibitor Treatment At Acute Phase of Spinal Cord Injury May Induce Neurite Outgrowth and Synaptogenesis.

PubMed

Devaux, Stephanie; Cizkova, Dasa; Mallah, Khalil; Karnoub, Melodie Anne; Laouby, Zahra; Kobeissy, Firas; Blasko, Juraj; Nataf, Serge; Pays, Laurent; Mériaux, Céline; Fournier, Isabelle; Salzet, Michel

2017-08-01

The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration/degeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we assessed in a rat SCI model the in vivo impact of RhoAi treatment administered in situ via alginate scaffold that was combined with FK506 delivery. The improved recovery of locomotion was detected only at the early postinjury time points, whereas after overall survival a dramatic increase of synaptic contacts on outgrowing neurites in affected segments was observed. We validate these results by in vivo proteomic studies along the spinal cord segments from tissue and secreted media analyses, confirming the increase of the synaptogenesis expression factors under RhoAi treatment. Taken together, we demonstrate that RhoAi treatment seems to be useful to stimulate neurite outgrowth in both in vitro as well in vivo environments. However, for in vivo experiments there is a need for sustained delivery regiment to facilitate axon regeneration and promote synaptic reconnections with appropriate target neurons also at chronic phase, which in turn may lead to higher assumption for functional improvement. © 2017 by The American Society for Biochemistry

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

Increased miR-124-3p in microglial exosomes following traumatic brain injury inhibits neuronal inflammation and contributes to neurite outgrowth via their transfer into neurons.

PubMed

Huang, Shan; Ge, Xintong; Yu, Jinwen; Han, Zhaoli; Yin, Zhenyu; Li, Ying; Chen, Fanglian; Wang, Haichen; Zhang, Jianning; Lei, Ping

2018-01-01

Neuronal inflammation is the characteristic pathologic change of acute neurologic impairment and chronic traumatic encephalopathy after traumatic brain injury (TBI). Inhibiting the excessive inflammatory response is essential for improving the neurologic outcome. To clarify the regulatory mechanism of microglial exosomes on neuronal inflammation in TBI, we focused on studying the impact of microglial exosomal miRNAs on injured neurons in this research. We used a repetitive (r)TBI mouse model and harvested the injured brain extracts from the acute to the chronic phase of TBI to treat cultured BV2 microglia in vitro The microglial exosomes were collected for miRNA microarray analysis, which showed that the expression level of miR-124-3p increased most apparently in the miRNAs. We found that miR-124-3p promoted the anti-inflamed M2 polarization in microglia, and microglial exosomal miR-124-3p inhibited neuronal inflammation in scratch-injured neurons. Further, the mammalian target of rapamycin (mTOR) signaling was implicated as being involved in the regulation of miR-124-3p by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Using the mTOR activator MHY1485 we confirmed that the inhibitory effect of exosomal miR-124-3p on neuronal inflammation was exerted by suppressing the activity of mTOR signaling. PDE4B was predicted to be the target gene of miR-124-3p by pathway analysis. We proved that it was directly targeted by miR-124-3p with a luciferase reporter assay. Using a PDE4B overexpressed lentivirus transfection system, we suggested that miR-124-3p suppressed the activity of mTOR signaling mainly through inhibiting the expression of PDE4B. In addition, exosomal miR-124-3p promoted neurite outgrowth after scratch injury, characterized by an increase on the number of neurite branches and total neurite length, and a decreased expression on RhoA and neurodegenerative proteins [Aβ-peptide and p-Tau]. It also improved the neurologic outcome and

Construction of a multi-functional extracellular matrix protein that increases number of N1E-115 neuroblast cells having neurites.

PubMed

Nakamura, Makiko; Mie, Masayasu; Mihara, Hisakazu; Nakamura, Makoto; Kobatake, Eiry

2009-10-01

An artificially designed fusion protein, which was designed to have strong cell adhesive activity and an active functional unit that enhances neuronal differentiation of mouse N1E-115 neuroblast cells, was developed. In this study, a laminin-1-derived IKVAV sequence, which stimulates neurite outgrowth in conditions of serum deprivation, was engineered and incorporated into an elastin-derived structural unit. The designed fusion protein also had a cell-adhesive RGD sequence derived from fibronectin. The resultant fusion protein could adsorb efficiently onto hydrophobic culture surfaces and showed cell adhesion activity similar to laminin. N1E-115 cells grown on the fusion protein exhibited more cells with neurites than cells grown on laminin-1. These results indicated that the constructed protein could retain properties of incorporated functional peptides and could provide effective signal transport. The strategy of designing multi-functional fusion proteins has the possibility for supporting current tissue engineering techniques. (c) 2009 Wiley Periodicals, Inc.

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

NASA Astrophysics Data System (ADS)

Wood, Matthew D.; Willits, Rebecca Kuntz

2009-08-01

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

Expression of Inapproptriate Cadherins in Human Breast Carcinomas

DTIC Science & Technology

2000-08-01

fibroblast growth factor receptor signaling. * We showed that cadherin 11 acts in a manner... fibroblast growth factor receptor signaling; and that cadherin 11 promotes epithelial cell motility in a manner similar to N-cadherin. 28 N-Cadherin...levels of E-cadherin; and that N- cadherin-dependent motility may be mediated by fibroblast growth factor receptor signaling. 14. SUBJECT TERMS

N-cadherin prodomain processing regulates synaptogenesis.

PubMed

Reinés, Analía; Bernier, Louis-Philippe; McAdam, Robyn; Belkaid, Wiam; Shan, Weisong; Koch, Alexander W; Séguéla, Philippe; Colman, David R; Dhaunchak, Ajit S

2012-05-02

Classical cadherins, which are adhesion molecules functioning at the CNS synapse, are synthesized as adhesively inactive precursor proteins in the endoplasmic reticulum (ER). Signal sequence and prodomain cleavage in the ER and Golgi apparatus, respectively, activates their adhesive properties. Here, we provide the first evidence for sorting of nonadhesive precursor N-cadherin (ProN) to the neuronal surface, where it coexists with adhesively competent mature N-cadherin (N-cad), generating a spectrum of adhesive strengths. In cultured hippocampal neurons, a high ProN/N-cad ratio downregulates synapse formation. Neurons expressing genetically engineered uncleavable ProN make markedly fewer synapses. The synapse number can be rescued to normality by depleting surface ProN levels through prodomain cleavage by an exogenous protease. Finally, prodomain processing is developmentally regulated in the rat hippocampus. We conclude that it is the ProN/N-cad ratio and not mature N-cad alone that is critical for regulation of adhesion during synaptogenesis.

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sindi, Ramya A., E-mail: ramya.sindi2010@my.ntu.ac

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 20 h before exposure to CPF or CPO for 2–8 h. 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 ofmore » axon-like processes compared to the control, respectively, retraction of neurites being observed within 2 h 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 2 h exposure and reduced levels of reactivity of the same antibody following 8 h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2 h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8 h 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. - Highlights: • Chlorpyrifos and chlorpyrifos oxon induced rapid neurite retraction in N2a cells. • This occurred following transient hyperphosphorylation of ERK 1/2. • It was concomitant

N-CADHERIN PRODOMAIN CLEAVAGE REGULATES SYNAPSE FORMATION IN VIVO

PubMed Central

Latefi, Nazlie S.; Pedraza, Liliana; Schohl, Anne; Li, Ziwei; Ruthazer, Edward S.

2009-01-01

Cadherins are initially synthesized bearing a prodomain that is thought to limit adhesion during early stages of biosynthesis. Functional cadherins lack this prodomain, raising the intriguing possibility that cells may utilize prodomain cleavage as a means to temporally or spatially regulate adhesion after delivery of cadherin to the cell surface. In support of this idea, immunostaining for the prodomain of zebrafish N-cadherin revealed enriched labeling at neuronal surfaces at the soma and along axonal processes. To determine whether post-translational cleavage of the prodomain affects synapse formation, we imaged Rohon-Beard cells in zebrafish embryos expressing GFP-tagged wild-type N-cadherin (NCAD-GFP) or a GFP-tagged N-cadherin mutant expressing an uncleavable prodomain (PRON-GFP) rendering it non-adhesive. NCAD-GFP accumulated at synaptic microdomains in a developmentally regulated manner, and its overexpression transiently accelerated synapse formation. PRON-GFP was much more diffusely distributed along the axon and its overexpression delayed synapse formation. Our results support the notion that N-cadherin serves to stabilize pre- to postsynaptic contacts early in synapse development and suggests that regulated cleavage of the N-cadherin prodomain may be a mechanism by which the kinetics of synaptogenesis are regulated. PMID:19365814

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

PubMed

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

2010-05-01

beta-Amyloid peptide (Abeta) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Abeta-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Abeta and on neurite outgrowth in PC12 cells were investigated. Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Abeta(1-42). Similar protective effects against Abeta(1-42) were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Abeta load was markedly diminished in the brain of those animals after treatment with piracetam. Abeta production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Abeta-induced mitochondrial dysfunction and Abeta-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Abeta on brain function.

Nanotechnology versus stem cell engineering: in vitro comparison of neurite inductive potentials.

PubMed

Morano, Michela; Wrobel, Sandra; Fregnan, Federica; Ziv-Polat, Ofra; Shahar, Abraham; Ratzka, Andreas; Grothe, Claudia; Geuna, Stefano; Haastert-Talini, Kirsten

2014-01-01

Innovative nerve conduits for peripheral nerve reconstruction are needed in order to specifically support peripheral nerve regeneration (PNR) whenever nerve autotransplantation is not an option. Specific support of PNR could be achieved by neurotrophic factor delivery within the nerve conduits via nanotechnology or stem cell engineering and transplantation. Here, we comparatively investigated the bioactivity of selected neurotrophic factors conjugated to iron oxide nanoparticles (np-NTFs) and of bone marrow-derived stem cells genetically engineered to overexpress those neurotrophic factors (NTF-BMSCs). The neurite outgrowth inductive activity was monitored in culture systems of adult and neonatal rat sensory dorsal root ganglion neurons as well as in the cell line from rat pheochromocytoma (PC-12) cell sympathetic culture model system. We demonstrate that np-NTFs reliably support numeric neurite outgrowth in all utilized culture models. In some aspects, especially with regard to their long-term bioactivity, np-NTFs are even superior to free NTFs. Engineered NTF-BMSCs proved to be less effective in induction of sensory neurite outgrowth but demonstrated an increased bioactivity in the PC-12 cell culture system. In contrast, primary nontransfected BMSCs were as effective as np-NTFs in sensory neurite induction and demonstrated an impairment of neuronal differentiation in the PC-12 cell system. Our results evidence that nanotechnology as used in our setup is superior over stem cell engineering when it comes to in vitro models for PNR. Furthermore, np-NTFs can easily be suspended in regenerative hydrogel matrix and could be delivered that way to nerve conduits for future in vivo studies and medical application.

Patterned cortical tension mediated by N-cadherin controls cell geometric order in the Drosophila eye

PubMed Central

Chan, Eunice HoYee; Chavadimane Shivakumar, Pruthvi; Clément, Raphaël; Laugier, Edith; Lenne, Pierre-François

2017-01-01

Adhesion molecules hold cells together but also couple cell membranes to a contractile actomyosin network, which limits the expansion of cell contacts. Despite their fundamental role in tissue morphogenesis and tissue homeostasis, how adhesion molecules control cell shapes and cell patterns in tissues remains unclear. Here we address this question in vivo using the Drosophila eye. We show that cone cell shapes depend little on adhesion bonds and mostly on contractile forces. However, N-cadherin has an indirect control on cell shape. At homotypic contacts, junctional N-cadherin bonds downregulate Myosin-II contractility. At heterotypic contacts with E-cadherin, unbound N-cadherin induces an asymmetric accumulation of Myosin-II, which leads to a highly contractile cell interface. Such differential regulation of contractility is essential for morphogenesis as loss of N-cadherin disrupts cell rearrangements. Our results establish a quantitative link between adhesion and contractility and reveal an unprecedented role of N-cadherin on cell shapes and cell arrangements. DOI: http://dx.doi.org/10.7554/eLife.22796.001 PMID:28537220

Quantifying Spiral Ganglion Neurite and Schwann Behavior on Micropatterned Polymer Substrates.

PubMed

Cheng, Elise L; Leigh, Braden; Guymon, C Allan; Hansen, Marlan R

2016-01-01

The first successful in vitro experiments on the cochlea were conducted in 1928 by Honor Fell (Fell, Arch Exp Zellforsch 7(1):69-81, 1928). Since then, techniques for culture of this tissue have been refined, and dissociated primary culture of the spiral ganglion has become a widely accepted in vitro model for studying nerve damage and regeneration in the cochlea. Additionally, patterned substrates have been developed that facilitate and direct neural outgrowth. A number of automated and semi-automated methods for quantifying this neurite outgrowth have been utilized in recent years (Zhang et al., J Neurosci Methods 160(1):149-162, 2007; Tapias et al., Neurobiol Dis 54:158-168, 2013). Here, we describe a method to study the effect of topographical cues on spiral ganglion neurite and Schwann cell alignment. We discuss our microfabrication process, characterization of pattern features, cell culture techniques for both spiral ganglion neurons and spiral ganglion Schwann cells. In addition, we describe protocols for reducing fibroblast count, immunocytochemistry, and methods for quantifying neurite and Schwann cell alignment.

Neurite Outgrowth and Neuroprotective Effects of Quercetin from Caesalpinia mimosoides Lamk. on Cultured P19-Derived Neurons

PubMed Central

Tangsaengvit, Napat; Kitphati, Worawan; Tadtong, Sarin; Bunyapraphatsara, Nuntavan; Nukoolkarn, Veena

2013-01-01

Quercetin has been isolated for the first time from ethyl acetate extract of Caesalpinia mimosoides Lamk. C. mimosoides Lamk. (Fabaceae) or Cha rueat (Thai name) is an indigenous plant found in mixed deciduous forest in northern and north-eastern parts of Thailand. Thai rural people consume its young shoots and leaves as a fresh vegetable, as well as it is used for medicinal purposes.The antioxidant capacity in terms of radical scavenging activity of quercetin was determined as IC50 of 3.18 ± 0.07 µg/mL, which was higher than that of Trolox and ascorbic acid (12.54 ± 0.89 and 10.52 ± 0.48 µg/mL, resp.). The suppressive effect of quercetin on both purified and cellular acetylcholinesterase (AChE) enzymes was investigated as IC50 56.84 ± 2.64 and 36.60 ± 2.78 µg/mL, respectively. In order to further investigate the protective ability of quercetin on neuronal cells, P19-derived neurons were used as a neuronal model in this study. As a result, quercetin at a very low dose of 1 nM enhanced survival and induced neurite outgrowth of P19-derived neurons. Furthermore, this flavonoid also possessed significant protection against oxidative stress induced by serum deprivation. Altogether, these findings suggest that quercetin is a multifunctional compound and promising valuable drugs candidate for the treatment of neurodegenerative disease. PMID:23840266

Automated quantification of neurite outgrowth orientation distributions on patterned surfaces

NASA Astrophysics Data System (ADS)

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

2014-08-01

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

Catenin-dependent cadherin function drives divisional segregation of spinal motor neurons.

PubMed

Bello, Sanusi M; Millo, Hadas; Rajebhosale, Manisha; Price, Stephen R

2012-01-11

Motor neurons that control limb movements are organized as a neuronal nucleus in the developing ventral horn of the spinal cord called the lateral motor column. Neuronal migration segregates motor neurons into distinct lateral and medial divisions within the lateral motor column that project axons to dorsal or ventral limb targets, respectively. This migratory phase is followed by an aggregation phase whereby motor neurons within a division that project to the same muscle cluster together. These later phases of motor neuron organization depend on limb-regulated differential cadherin expression within motor neurons. Initially, all motor neurons display the same cadherin expression profile, which coincides with the migratory phase of motor neuron segregation. Here, we show that this early, pan-motor neuron cadherin function drives the divisional segregation of spinal motor neurons in the chicken embryo by controlling motor neuron migration. We manipulated pan-motor neuron cadherin function through dissociation of cadherin binding to their intracellular partners. We found that of the major intracellular transducers of cadherin signaling, γ-catenin and α-catenin predominate in the lateral motor column. In vivo manipulations that uncouple cadherin-catenin binding disrupt divisional segregation via deficits in motor neuron migration. Additionally, reduction of the expression of cadherin-7, a cadherin predominantly expressed in motor neurons only during their migration, also perturbs divisional segregation. Our results show that γ-catenin-dependent cadherin function is required for spinal motor neuron migration and divisional segregation and suggest a prolonged role for cadherin expression in all phases of motor neuron organization.

Suppression of tumorigenicity by plakoglobin: an augmenting effect of N-cadherin.

PubMed

Simcha, I; Geiger, B; Yehuda-Levenberg, S; Salomon, D; Ben-Ze'ev, A

1996-04-01

Plakoglobin is a major component of the submembranal plaque of adherens junctions and desmosomes in mammalian cells. It is closely related to the Drosophila segment polarity gene armadillo which has a role in the transduction of transmembrane signals that regulate cell fate. Like its close homologue beta-catenin, plakoglobin can associate with the product of the tumor suppressor gene APC that is linked to human colon cancer. We have studied the effect of plakoglobin overexpression, and the cooperation between plakoglobin and N-cadherin, on the morphology and tumorigenic ability of cells either lacking, or expressing cadherin and alpha- and beta-catenin. Overexpression of plakoglobin in SV40-transformed 3T3 (SVT2) cells suppressed the tumorigenicity of the cells in syngeneic mice. Transfection with N-cadherin conferred an epithelial phenotype on the cell culture, but had no significant effect on the tumorigenicity of the cells. Cotransfection of plakoglobin and N-cadherin into SVT2 cells, however, was considerably more effective in tumor suppression than plakoglobin overexpression alone. Finally, transfection of plakoglobin into a human renal carcinoma cell line that expresses neither cadherins nor plakoglobin, or alpha-and beta-catenin, resulted in a dose-dependent suppression of tumor formation by these cells in nude mice. Plakoglobin, in these cells, did not exhibit junctional localization and was diffusely distributed in the cytoplasm, with a significant amount of the protein also localized in the nucleus. The results suggest that plakoglobin can efficiently suppress the tumorigenicity of cells in the presence of, or independently of the cadherin-catenin complex.

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea.

PubMed

Huang, Lin-Chien; Thorne, Peter R; Housley, Gary D; Montgomery, Johanna M

2007-08-01

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.

Unconventional Cadherin Localization in Honey Bee Gonads Revealed Through Domain-Specific Apis mellifera E- and N-Cadherin Antibodies Indicates Alternative Functions.

PubMed

Florecki, Mônica M; Hartfelder, Klaus

2012-11-22

As key factors in intercellular adhesion processes, cadherins play important roles in a plethora of developmental processes, including gametogenesis. In a previous study on cadherin localization in the gonads of honey bees, performed with heterologous pan-cadherin antibodies, we detected these proteins as (i) associated with cell membranes, (ii) as homogeneously distributed throughout the cytoplasm, and (iii) as nuclear foci in both somatic and germline cells, raising the possibility of alternative functions. To further investigate such unusual intracellular cadherin localization we produced specific antibodies against the N- and C-terminal domains of honey bee N- and E-cadherin. A 160 kDa protein was recognized by the E-cadherin antibodies as well as one of approximately 300 kDa from those raised against N-cadherin. In gonad preparations, both proteins were detected as dispersed throughout the cytoplasm and as nuclear foci in both germline and somatic cells of queen and worker ovarioles, as well as in the testioles of drones. This leads us to infer that cadherins may indeed be involved in certain signaling pathways and/or transcriptional regulation during gametogenesis. In late oogenesis stages, immunolabeling for both proteins was observed at the cell cortex, in conformity with a role in cell adhesion. In testioles, E-cadherin was seen in co-localization with fusomes, indicating a possible role in cyst organization. Taken together, the distribution of N- and E-cadherins in honey bee gonads is suggestive of alternative roles for cadherins in gametogenesis of both sexes.

Salubrinal inhibits the expression of proteoglycans and favors neurite outgrowth from cortical neurons in vitro.

PubMed

Barreda-Manso, M Asunción; Yanguas-Casás, Natalia; Nieto-Sampedro, Manuel; Romero-Ramírez, Lorenzo

2015-07-01

After CNS injury, astrocytes and mesenchymal cells attempt to restore the disrupted glia limitans by secreting proteoglycans and extracellular matrix proteins (ECMs), forming the so-called glial scar. Although the glial scar is important in sealing the lesion, it is also a physical and functional barrier that prevents axonal regeneration. The synthesis of secretory proteins in the RER is under the control of the initiation factor of translation eIF2α. Inhibiting the synthesis of secretory proteins by increasing the phosphorylation of eIF2α, might be a pharmacologically efficient way of reducing proteoglycans and other profibrotic proteins present in the glial scar. Salubrinal, a neuroprotective drug, decreased the expression and secretion of proteoglycans and other profibrotic proteins induced by EGF or TGFβ, maintaining eIF2α phosphorylated. Besides, Salubrinal also reduced the transcription of proteoglycans and other profibrotic proteins, suggesting that it induced the degradation of non-translated mRNA. In a model in vitro of the glial scar, cortical neurons grown on cocultures of astrocytes and fibroblasts with TGFβ treated with Salubrinal, showed increased neurite outgrowth compared to untreated cells. Our results suggest that Salubrinal may be considered of therapeutic value facilitating axonal regeneration, by reducing overproduction and secretion of proteoglycans and profibrotic protein inhibitors of axonal growth. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro neurotoxic effects of 1 GeV/n iron particles assessed in retinal explants.

PubMed

Vazquez, M E; Kirk, E

2000-01-01

The heavy ion component of the cosmic radiation remains problematic to the assessment of risk in manned space flight. The biological effectiveness of HZE particles has yet to be established, particularly with regard to nervous tissue. Using heavy ions accelerated at the AGS of Brookhaven National Laboratory, we study the neurotoxic effects of iron particles. We exposed retinal explants, taken from chick embryos, to determine the dose response relationships for neurite outgrowth. Morphometric techniques were used to evaluate the in vitro effects of 1 GeV/a iron particles (LET 148 keV/micrometer). Iron particles produced a dose-dependent reduction of neurite outgrowth with a maximal effect achieved with a dose of 100 cGy. Doses as low as 10-50 cGy were able to induce reductions of the neurite outgrowth as compared to the control group. Neurite generation is a more sensitive parameter than neurite elongation, suggesting different mechanism of radiation damage in our model. These results showed that low doses/fluences of iron particles could impair the retinal ganglion cells' capacity to generate neurites indicating the highly neurotoxic capability of this heavy charged particle.

IMPACT Is a Developmentally Regulated Protein in Neurons That Opposes the Eukaryotic Initiation Factor 2α Kinase GCN2 in the modulation of Neurite Outgrowth*

PubMed Central

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

2013-01-01

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. PMID:23447528

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.

Deep analysis of N-cadherin/ADH-1 interaction: a computational survey.

PubMed

Eslami, Mahboobeh; Nezafat, Navid; Khajeh, Sahar; Mostafavi-Pour, Zohreh; Bagheri Novir, Samaneh; Negahdaripour, Manica; Ghasemi, Younes; Razban, Vahid

2018-01-19

Due to the considerable role of N-cadherin in cancer metastasis, tumor growth, and progression, inhibition of this protein has been highly regarded in recent years. Although ADH-1 has been known as an appropriate inhibitor of N-cadherin in clinical trials, its chemical nature and binding mode with N-cadherin have not been precisely specified yet. Accordingly, in this study, quantum mechanics calculations were used to investigate the chemical nature of ADH-1. These calculations clarify the molecular properties of ADH-1 and determine its reactive sites. Based on the results, the oxygen atoms are suitable for electrophilic reactivity, while the hydrogen atoms that are connected to nitrogen atoms are the favorite sites for nucleophilic reactivity. The higher electronegativity of the oxygen atoms makes them the most reactive portions in this molecule. Molecular docking and molecular dynamics (MD) simulation have also been applied to specify the binding mode of ADH-1 with N-cadherin and determine the important residues of N-cadherin involving in the interaction with ADH-1. Moreover, the verified model by MD simulation has been studied to extract the free energy value and find driving forces. These calculations and molecular electrostatic potential map of ADH-1 indicated that hydrophobic and electrostatic interactions are almost equally involved in the implantation of ADH-1 in the N-cadherin binding site. The presented results not only enable a closer examination of N-cadherin in complex with ADH-1 molecule, but also are very beneficial in designing new inhibitors for N-cadherin and can help to save time and cost in this field.

Unconventional Cadherin Localization in Honey Bee Gonads Revealed Through Domain-Specific Apis mellifera E- and N-Cadherin Antibodies Indicates Alternative Functions

PubMed Central

Florecki, Mônica M.; Hartfelder, Klaus

2012-01-01

As key factors in intercellular adhesion processes, cadherins play important roles in a plethora of developmental processes, including gametogenesis. In a previous study on cadherin localization in the gonads of honey bees, performed with heterologous pan-cadherin antibodies, we detected these proteins as (i) associated with cell membranes, (ii) as homogeneously distributed throughout the cytoplasm, and (iii) as nuclear foci in both somatic and germline cells, raising the possibility of alternative functions. To further investigate such unusual intracellular cadherin localization we produced specific antibodies against the N- and C-terminal domains of honey bee N- and E-cadherin. A 160 kDa protein was recognized by the E-cadherin antibodies as well as one of approximately 300 kDa from those raised against N-cadherin. In gonad preparations, both proteins were detected as dispersed throughout the cytoplasm and as nuclear foci in both germline and somatic cells of queen and worker ovarioles, as well as in the testioles of drones. This leads us to infer that cadherins may indeed be involved in certain signaling pathways and/or transcriptional regulation during gametogenesis. In late oogenesis stages, immunolabeling for both proteins was observed at the cell cortex, in conformity with a role in cell adhesion. In testioles, E-cadherin was seen in co-localization with fusomes, indicating a possible role in cyst organization. Taken together, the distribution of N- and E-cadherins in honey bee gonads is suggestive of alternative roles for cadherins in gametogenesis of both sexes. PMID:26466735

PDGF controls contact inhibition of locomotion by regulating N-cadherin during neural crest migration.

PubMed

Bahm, Isabel; Barriga, Elias H; Frolov, Antonina; Theveneau, Eric; Frankel, Paul; Mayor, Roberto

2017-07-01

A fundamental property of neural crest (NC) migration is contact inhibition of locomotion (CIL), a process by which cells change their direction of migration upon cell contact. CIL has been proven to be essential for NC migration in amphibians and zebrafish by controlling cell polarity in a cell contact-dependent manner. Cell contact during CIL requires the participation of the cell adhesion molecule N-cadherin, which starts to be expressed by NC cells as a consequence of the switch between E- and N-cadherins during epithelial-to-mesenchymal transition (EMT). However, the mechanism that controls the upregulation of N-cadherin remains unknown. Here, we show that platelet-derived growth factor receptor alpha (PDGFRα) and its ligand platelet-derived growth factor A (PDGF-A) are co-expressed in migrating cranial NC. Inhibition of PDGF-A/PDGFRα blocks NC migration by inhibiting N-cadherin and, consequently, impairing CIL. Moreover, we identify phosphatidylinositol-3-kinase (PI3K)/AKT as a downstream effector of the PDGFRα cellular response during CIL. Our results lead us to propose PDGF-A/PDGFRα signalling as a tissue-autonomous regulator of CIL by controlling N-cadherin upregulation during EMT. Finally, we show that once NC cells have undergone EMT, the same PDGF-A/PDGFRα works as an NC chemoattractant, guiding their directional migration. © 2017. Published by The Company of Biologists Ltd.

M-cadherin and its sisters in development of striated muscle.

PubMed

Kaufmann, U; Martin, B; Link, D; Witt, K; Zeitler, R; Reinhard, S; Starzinski-Powitz, A

1999-04-01

Cadherins are calcium-dependent, transmembrane intercellular adhesion proteins with morphoregulatory functions in the development and maintenance of tissues. In the development of striated muscle, the expression and function of mainly M-, N-, and R-cadherin has been studied so far. While these three cadherins are expressed in skeletal muscle cells, of these only N-cadherin is expressed in cardiac muscle. In this review, M-, N-, and R-cadherin are discussed as important players in the terminal differentiation and possibly also in the commitment of skeletal muscle cells. Furthermore, reports are described which evaluate the essential role of N-cadherin in the formation of heart tissue.

Piperine-like alkamides from Piper nigrum induce BDNF promoter and promote neurite outgrowth in Neuro-2a cells.

PubMed

Yun, Young Sook; Noda, Sachie; Takahashi, Shigeru; Takahashi, Yuji; Inoue, Hideshi

2018-01-01

Black pepper (Piper nigrum) contains a variety of alkamides. Among them, piperine has been reported to have antidepressant-like effects in chronically stressed mice, but little is known about the biological activity of other alkamides. In this study, we investigated the effects of alkamides from white pepper (P. nigrum) on neuronal cells. Twelve alkamides were isolated from white pepper MeOH extracts, and their chemical structures were identified by NMR and MS analyses. The compounds were subjected to assays using the luciferase-reporter gene under the control of the BDNF promoter or cAMP response element in mouse neuroblastoma Neuro-2a cells. In both assays, marked reporter-inducing activity was observed for piperine (1), piperettine (2) and piperylin (7), all of which have in common an (E)-5-(buta-1,3-dien-1-yl)benzo[d] [1, 3] dioxole moiety. Piperettine (2) and piperylin (7) tended to increase endogenous BDNF protein levels. Furthermore, piperylin (7) promoted retinoic acid-induced neurite outgrowth. These results suggest that piperylin (7), or analogues thereof, may have a beneficial effect on disorders associated with dysregulation of BDNF expression, such as depression.

Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells.

PubMed

Tang, Li-li; Wang, Rui; Tang, Xi-can

2005-06-01

To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). After being treated with 10 micromol/L HupA, neurite outgrowth of PC12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. AChE activity, mRNA and protein expression were measured by the Ellman method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. Treatment of PC12 cells with 10 micromol/L HupA for 48 h markedly increased the number of neurite-bearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10 micromol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2 h exposure of the astrocytes to 10 micromol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.

Dilysine motifs in exon 2b of SMN protein mediate binding to the COPI vesicle protein α-COP and neurite outgrowth in a cell culture model of spinal muscular atrophy.

PubMed

Custer, Sara K; Todd, Adrian G; Singh, Natalia N; Androphy, Elliot J

2013-10-15

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that stems from low levels of survival of motor neuron (SMN) protein. The processes that cause motor neurons and muscle cells to become dysfunctional are incompletely understood. We are interested in neuromuscular homeostasis and the stresses put upon that system by loss of SMN. We recently reported that α-COP, a member of the coatomer complex of coat protein I (COPI) vesicles, is an SMN-binding partner, implicating this protein complex in normal SMN function. To investigate the functional significance of the interaction between α-COP and SMN, we constructed an inducible NSC-34 cell culture system to model the consequences of SMN depletion and find that depletion of SMN protein results in shortened neurites. Heterologous expression of human SMN, and interestingly over-expression of α-COP, restores normal neurite length and morphology. Mutagenesis of the canonical COPI dilysine motifs in exon 2b results in failure to bind to α-COP and abrogates the ability of human SMN to restore neurite outgrowth in SMN-depleted motor neuron-like NSC-34 cells. We conclude that the interaction between SMN and α-COP serves an important function in the growth and maintenance of motor neuron processes and may play a significant role in the pathogenesis of SMA.

Laminin promotes neuritic regeneration from cultured peripheral and central neurons

PubMed Central

1983-01-01

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co- purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin. PMID:6643580

Neuroglian and FasciclinII can promote neurite outgrowth via the FGF receptor Heartless.

PubMed

Forni, John J; Romani, Susana; Doherty, Patrick; Tear, Guy

2004-06-01

To further investigate the role of the Drosophila cell adhesion molecules (CAMs), we have developed an in vitro assay that allows us to test the contribution individual CAMs make to promote outgrowth of specific Drosophila neurons. The extension of primary cultured neurons on a substrate of purified recombinant CAM is measured. We show that both FasciclinII and Neuroglian are able to promote outgrowth of FasciclinII or Neuroglian expressing neurons, respectively. Furthermore, this growth promotion activity is provided when the CAMs are presented both in a substrate bound or soluble form. We also show that the signal provided by the CAMs acts via the Heartless fibroblast growth factor receptor (FGFR) as outgrowth is reduced to basal levels in the presence of an FGFR inhibitor or if Heartless function is missing from the neurons. Copyright 2004 Elsevier Inc.

Prognostic Potential of N-Cadherin in Oral Squamous Cell Carcinoma via Immunohistochemical Methods.

PubMed

Chandolia, Betina; Rajliwal, Jai Parkash; Bajpai, Manas; Arora, Manika

2017-08-01

To assess the prognostic potential for N-cadherin in oral squamous cell carcinoma and oral epithelial dysplasia. Across-sectional study, analytical study. Maharishi Markandeshwar College of Dental Science Research (MMCDSR), Ambala, India, from 2011 to 2014. Immunohistochemistry was used to observe the N-cadherin expression in 100 cases having epithelium with normal oral mucosa, oral epithelial dysplastic lesions and oral squamous cell carcinoma (OSCC). For statistical significance, SPSS 13.0 was used to calculate the data by Mann-Whitney and Kruskal-Wallis tests. In OSCC, N-cadherin expression was more evident than in oral epithelial dysplasia followed by the normal oral epithelium that did not show any dysplastic changes (p=0.001). Conversely, N-cadherin expression was not significant among the histological grade of OSCC. N-cadherin can be used as a potential biomarker for early diagnosis of OSCC. However, the N-cadherin expression did not show any correlation with the histological grade of OSCC.

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

PubMed

Ashcroft, M; Stephens, R M; Hallberg, B; Downward, J; Kaplan, D R

1999-08-12

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Epidermal growth factor receptor and integrins control force-dependent vinculin recruitment to E-cadherin junctions.

PubMed

Sehgal, Poonam; Kong, Xinyu; Wu, Jun; Sunyer, Raimon; Trepat, Xavier; Leckband, Deborah

2018-03-20

This study reports novel findings that link E-cadherin (also known as CDH1)-mediated force-transduction signaling to vinculin targeting to intercellular junctions via epidermal growth factor receptor (EGFR) and integrins. These results build on previous findings that demonstrated that mechanically perturbed E-cadherin receptors activate phosphoinositide 3-kinase and downstream integrins in an EGFR-dependent manner. Results of this study show that this EGFR-mediated kinase cascade controls the force-dependent recruitment of vinculin to stressed E-cadherin complexes - a key early signature of cadherin-based mechanotransduction. Vinculin targeting requires its phosphorylation at tyrosine 822 by Abl family kinases (hereafter Abl), but the origin of force-dependent Abl activation had not been identified. We now present evidence that integrin activation, which is downstream of EGFR signaling, controls Abl activation, thus linking E-cadherin to Abl through a mechanosensitive signaling network. These findings place EGFR and integrins at the center of a positive-feedback loop, through which force-activated E-cadherin signals regulate vinculin recruitment to cadherin complexes in response to increased intercellular tension.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase.

PubMed

Spencer, Michael L; Shao, Haipeng; Andres, Douglas A

2002-06-07

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.

N-cadherin expression in palisade nerve endings of rat vellus hairs.

PubMed

Kaidoh, Toshiyuki; Inoué, Takao

2008-02-01

Palisade nerve endings (PNs) are mechanoreceptors around vellus hairs of mammals. Each lanceolate nerve ending (LN) of the PN is characterized by a sensory nerve ending symmetrically sandwiched by two processes of type II terminal Schwann cells (tSCIIs). However, the molecular mechanisms underlying the structural organization of the PN are poorly understood. Electron microscopy showed that adherens junctions appeared to adhere to the sensory nerve ending and tSCII processes, so we examined the location of the N-cadherin adhesion system in PNs of rat vellus hairs by using immunoelectron microscopy. N-cadherin localized near both ends of the cell boundary between sensory nerve ending and tSCII processes, which corresponded to the sites of adherens junctions. We further found cadherin-associated proteins, alpha- and beta-catenins, at the linings of adherens junctions. Three-dimensional reconstruction of immunoelectron microscopic serial thin sections showed four linear arrays of N-cadherin arranged longitudinally along the LN beneath the four longitudinal borders of two tSCII processes. In contrast, sensory nerve fibers just proximal to the LNs formed common unmyelinated nerve fibers, in which N-cadherin was located mainly at the mesaxon of type I terminal Schwann cells (tSCIs). These results suggest that the four linear arrays of N-cadherin-mediated junctions adhere the sensory nerve ending and tSCII processes side by side to form the characteristic structure of the LN, and the structural differences between the LNs and the proximal unmyelinated nerve fibers possibly are due to the difference in the pattern of N-cadherin expression between sensory nerve endings and tSCII or tSCI processes. (c) 2007 Wiley-Liss, Inc.

Microtubule-Actin Crosslinking Factor 1 Is Required for Dendritic Arborization and Axon Outgrowth in the Developing Brain.

PubMed

Ka, Minhan; Kim, Woo-Yang

2016-11-01

Dendritic arborization and axon outgrowth are critical steps in the establishment of neural connectivity in the developing brain. Changes in the connectivity underlie cognitive dysfunction in neurodevelopmental disorders. However, molecules and associated mechanisms that play important roles in dendritic and axon outgrowth in the brain are only partially understood. Here, we show that microtubule-actin crosslinking factor 1 (MACF1) regulates dendritic arborization and axon outgrowth of developing pyramidal neurons by arranging cytoskeleton components and mediating GSK-3 signaling. MACF1 deletion using conditional mutant mice and in utero gene transfer in the developing brain markedly decreased dendritic branching of cortical and hippocampal pyramidal neurons. MACF1-deficient neurons showed reduced density and aberrant morphology of dendritic spines. Also, loss of MACF1 impaired the elongation of callosal axons in the brain. Actin and microtubule arrangement appeared abnormal in MACF1-deficient neurites. Finally, we found that GSK-3 is associated with MACF1-controlled dendritic differentiation. Our findings demonstrate a novel role for MACF1 in neurite differentiation that is critical to the creation of neuronal connectivity in the developing brain.

FAT1 cadherin acts upstream of Hippo signalling through TAZ to regulate neuronal differentiation.

PubMed

Ahmed, Abdulrzag F; de Bock, Charles E; Lincz, Lisa F; Pundavela, Jay; Zouikr, Ihssane; Sontag, Estelle; Hondermarck, Hubert; Thorne, Rick F

2015-12-01

The Hippo pathway is emerging as a critical nexus that balances self-renewal of progenitors against differentiation; however, upstream elements in vertebrate Hippo signalling are poorly understood. High expression of Fat1 cadherin within the developing neuroepithelium and the manifestation of severe neurological phenotypes in Fat1-knockout mice suggest roles in neurogenesis. Using the SH-SY5Y model of neuronal differentiation and employing gene silencing techniques, we show that FAT1 acts to control neurite outgrowth, also driving cells towards terminal differentiation via inhibitory effects on proliferation. FAT1 actions were shown to be mediated through Hippo signalling where it activated core Hippo kinase components and antagonised functions of the Hippo effector TAZ. Suppression of FAT1 promoted the nucleocytoplasmic shuttling of TAZ leading to enhanced transcription of the Hippo target gene CTGF together with accompanying increases in nuclear levels of Smad3. Silencing of TAZ reversed the effects of FAT1 depletion thus connecting inactivation of TAZ-TGFbeta signalling with Hippo signalling mediated through FAT1. These findings establish FAT1 as a new upstream Hippo element regulating early stages of differentiation in neuronal cells.

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

PubMed Central

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

2010-01-01

Background and purpose: β-Amyloid peptide (Aβ) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. Experimental approach: We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Aβ-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Aβ and on neurite outgrowth in PC12 cells were investigated. Key results: Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Aβ1-42. Similar protective effects against Aβ1-42 were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Aβ load was markedly diminished in the brain of those animals after treatment with piracetam. Aβ production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Aβ-induced mitochondrial dysfunction and Aβ-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Conclusion and implications: Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Aβ on brain function. This article is commented on by Moncada, pp. 217–219 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00706.x and to view related papers by Pravdic et al. and Puerta et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00698.x and http://dx.doi.org/10.1111/j

δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions*

PubMed Central

Yuan, Li; Seong, Eunju; Beuscher, James L.; Arikkath, Jyothi

2015-01-01

The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome. PMID:25724647

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

PubMed Central

Powrozek, Teresa A.; Olson, Eric C.

2012-01-01

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 (400mg/dL) ethanol for either 4 or 24 hrs 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. PMID:22840816

Precursor N-cadherin mediates glial cell line-derived neurotrophic factor-promoted human malignant glioma

PubMed Central

Zhu, Shuang; Zhang, Baole; Qin, Yuxia; Yao, Ruiqin; Zhou, Hao; Gao, Dian Shuai

2017-01-01

As the most prevalent primary brain tumor, gliomas are highly metastatic, invasive and are characteristic of high levels of glial cell-line derived neurotrophic factor (GDNF). GDNF is an important factor for invasive glioma cell growth; however, the underlying mechanism involved is unclear. In this study, we affirm a significantly higher expression of the precursor of N-cadherin (proN-cadherin) in most gliomas compared with normal brain tissues. Our findings reveal that GDNF interacts with the extracellular domain of proN-cadherin, which suggests that proN-cadherin mediates GDNF-induced glioma cell migration and invasion. We hypothesize that proN-cadherin might cause homotypic adhesion loss within neighboring cells and at the same time promote heterotypic adhesion within the extracellular matrix (ECM) through a certain mechanism. This study also demonstrates that the interaction between GDNF and proN-cadherin activates specific intracellular signaling pathways; furthermore, GDNF promoted the secretion of matrix metalloproteinase-9 (MMP-9), which degrades the ECM via proN-cadherin. To reach the future goal of developing novel therapies of glioma, this study, reveals a unique mechanism of glioma cell migration and invasion. PMID:28212546

Precursor N-cadherin mediates glial cell line-derived neurotrophic factor-promoted human malignant glioma.

PubMed

Xiong, Ye; Liu, Liyun; Zhu, Shuang; Zhang, Baole; Qin, Yuxia; Yao, Ruiqin; Zhou, Hao; Gao, Dian Shuai

2017-04-11

As the most prevalent primary brain tumor, gliomas are highly metastatic, invasive and are characteristic of high levels of glial cell-line derived neurotrophic factor (GDNF). GDNF is an important factor for invasive glioma cell growth; however, the underlying mechanism involved is unclear. In this study, we affirm a significantly higher expression of the precursor of N-cadherin (proN-cadherin) in most gliomas compared with normal brain tissues. Our findings reveal that GDNF interacts with the extracellular domain of proN-cadherin, which suggests that proN-cadherin mediates GDNF-induced glioma cell migration and invasion. We hypothesize that proN-cadherin might cause homotypic adhesion loss within neighboring cells and at the same time promote heterotypic adhesion within the extracellular matrix (ECM) through a certain mechanism. This study also demonstrates that the interaction between GDNF and proN-cadherin activates specific intracellular signaling pathways; furthermore, GDNF promoted the secretion of matrix metalloproteinase-9 (MMP-9), which degrades the ECM via proN-cadherin. To reach the future goal of developing novel therapies of glioma, this study, reveals a unique mechanism of glioma cell migration and invasion.

Rab coupling protein mediated endosomal recycling of N-cadherin influences cell motility.

PubMed

Lindsay, Andrew J; McCaffrey, Mary W

2017-12-01

Rab coupling protein (RCP) is a Rab GTPase effector that functions in endosomal recycling. The RCP gene is frequently amplified in breast cancer, leading to increased cancer aggressiveness. Furthermore, RCP enhances the motility of ovarian cancer cells by coordinating the recycling of α5β1 integrin and EGF receptor to the leading edge of migrating cells. Here we report that RCP also influences the motility of lung adenocarcinoma cells. Knockdown of RCP inhibits the motility of A549 cells in 2D and 3D migration assays, while its overexpression enhances migration in these assays. Depletion of RCP leads to a reduction in N-cadherin protein levels, which could be restored with lysosomal inhibitors. Trafficking assays revealed that RCP knockdown inhibits the return of endocytosed N-cadherin to the cell surface. We propose that RCP regulates the endosomal recycling of N-cadherin, and in its absence N-cadherin is diverted to the degradative pathway. The increased aggressiveness of tumour cells that overexpress RCP may be due to biased recycling of N-cadherin in metastatic cancer cells.

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

PubMed Central

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

2013-01-01

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

Recruitment of β-Catenin to N-Cadherin Is Necessary for Smooth Muscle Contraction*

PubMed Central

Wang, Tao; Wang, Ruping; Cleary, Rachel A.; Gannon, Olivia J.; Tang, Dale D.

2015-01-01

β-Catenin is a key component that connects transmembrane cadherin with the actin cytoskeleton at the cell-cell interface. However, the role of the β-catenin/cadherin interaction in smooth muscle has not been well characterized. Here stimulation with acetylcholine promoted the recruitment of β-catenin to N-cadherin in smooth muscle cells/tissues. Knockdown of β-catenin by lentivirus-mediated shRNA attenuated smooth muscle contraction. Nevertheless, myosin light chain phosphorylation at Ser-19 and actin polymerization in response to contractile activation were not reduced by β-catenin knockdown. In addition, the expression of the β-catenin armadillo domain disrupted the recruitment of β-catenin to N-cadherin. Force development, but not myosin light chain phosphorylation and actin polymerization, was reduced by the expression of the β-catenin armadillo domain. Furthermore, actin polymerization and microtubules have been implicated in intracellular trafficking. In this study, the treatment with the inhibitor latrunculin A diminished the interaction of β-catenin with N-cadherin in smooth muscle. In contrast, the exposure of smooth muscle to the microtubule depolymerizer nocodazole did not affect the protein-protein interaction. Together, these findings suggest that smooth muscle contraction is mediated by the recruitment of β-catenin to N-cadherin, which may facilitate intercellular mechanotransduction. The association of β-catenin with N-cadherin is regulated by actin polymerization during contractile activation. PMID:25713069

High butyric acid amounts induce oxidative stress, alter calcium homeostasis, and cause neurite retraction in nerve growth factor-treated PC12 cells.

PubMed

Cueno, Marni E; Kamio, Noriaki; Seki, Keisuke; Kurita-Ochiai, Tomoko; Ochiai, Kuniyasu

2015-07-01

Butyric acid (BA) is a common secondary metabolite by-product produced by oral pathogenic bacteria and is detected in high amounts in the gingival tissue of patients with periodontal disease. Previous works have demonstrated that BA can cause oxidative stress in various cell types; however, this was never explored using neuronal cells. Here, we exposed nerve growth factor (NGF)-treated PC1(2) cells to varying BA concentrations (0.5, 1.0, 5.0 mM). We measured total heme, H(2)O(2), catalase, and calcium levels through biochemical assays and visualized the neurite outgrowth after BA treatment. Similarly, we determined the effects of other common periodontal short-chain fatty acids (SCFAs) on neurite outgrowth for comparison. We found that high (1.0 and 5.0 mM) BA concentrations induced oxidative stress and altered calcium homeostasis, whereas low (0.5 mM) BA concentration had no significant effect. Moreover, compared to other SCFAs, we established that only BA was able to induce neurite retraction.

δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions.

PubMed

Yuan, Li; Seong, Eunju; Beuscher, James L; Arikkath, Jyothi

2015-04-24

The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Microtubule-Actin Crosslinking Factor 1 is required for dendritic arborization and axon outgrowth in the developing brain

PubMed Central

Ka, Minhan; Kim, Woo-Yang

2015-01-01

Dendritic arborization and axon outgrowth are critical steps in the establishment of neural connectivity in the developing brain. Changes in the connectivity underlie cognitive dysfunction in neurodevelopmental disorders. However, molecules and associated mechanisms that play important roles in dendritic and axon outgrowth in the brain are only partially understood. Here, we show that Microtubule-Actin Crosslinking Factor 1 (MACF1) regulates dendritic arborization and axon outgrowth of developing pyramidal neurons by arranging cytoskeleton components and mediating GSK-3 signaling. MACF1 deletion using conditional mutant mice and in utero gene transfer in the developing brain markedly decreased dendritic branching of cortical and hippocampal pyramidal neurons. MACF1-deficient neurons showed reduced density and aberrant morphology of dendritic spines. Also, loss of MACF1 impaired the elongation of callosal axons in the brain. Actin and microtubule arrangement appeared abnormal in MACF1-deficient neurites. Finally, we found that GSK-3 is associated with MACF1-controlled dendritic differentiation. Our findings demonstrate a novel role for MACF1 in neurite differentiation that is critical to the creation of neuronal connectivity in the developing brain. PMID:26526844

Effects of proinflammatory cytokines on axonal outgrowth from adult rat lumbar dorsal root ganglia using a novel three-dimensional culture system.

PubMed

Kim, Hyunchul; W Caspar, Tyler; Shah, Sameer B; Hsieh, Adam H

2015-08-01

Degeneration of the intervertebral disc is often associated with low back pain and increased infiltration of nerve fibers originating from dorsal root ganglia (DRG). The degenerated disc is also characterized by the presence of proinflammatory cytokines, which may influence axonal outgrowth. Toward an improved understanding of the growth of DRG neurons into compliant extracellular matrices, we developed a novel experimental system to measure axonal outgrowth of adult rat lumbar DRG neurons within three-dimensional (3D) collagen hydrogels and used this system to examine the effects of interleukin 1β (IL-1β) and tumor necrosis factor (TNF)-α treatment. The aim was to investigate the effects of proinflammatory cytokines on 3D neuronal growth into collagen matrices. This was an in vitro study of neurite outgrowth from adult rat lumbar DRG into collagen gels in response to IL-1β and TNF-α. Lumbar DRG were obtained from adult Sprague Dawley rats, bisected to expose cell bodies and placed onto collagen gel constructs prepared in 24-well Transwell inserts. Dorsal root ganglia were then treated with nerve growth factor (NGF)-free Neurobasal media (negative control) or NGF-supplemented media containing 0, 1, and 10 ng/mL of IL-1β and TNF-α. After 7 days, collagen gel-DRG constructs were immunostained for phosphorylated neurofilament, an axonal marker. Simple Neurite Tracer (Fiji/ImageJ) was used to quantify 3D axonal outgrowth from confocal image stacks. Data were analyzed using one-way analysis of variance, with Tukey HSD post hoc correction at a level of p<.05. Immunostaining showed robust axonal outgrowth into collagen gels from all NGF-treated DRG. The negative control demonstrated very few and short neurites. Tumor necrosis factor-α (1 and 10 ng/mL) significantly inhibited axonal outgrowth compared with NGF-only media (p<.026 and p<.02, respectively). After IL-1β treatment, average axon length was 10% lower at 1 ng/mL and 7.5% higher at 10 ng/mL, but these

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

PubMed Central

Dias, Ana M.; Oliveira, Patrícia; Cabral, Joana; Seruca, Raquel; Oliveira, Carla; Morgado-Díaz, José Andrés; Reis, Celso A.; Pinho, Salomé S.

2013-01-01

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications. PMID:24282611

The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

PubMed Central

Balsamo, Janne; Arregui, Carlos; Leung, TinChung; Lilien, Jack

1998-01-01

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion. PMID:9786960

Transforming growth factor β induces bone marrow mesenchymal stem cell migration via noncanonical signals and N-cadherin.

PubMed

Dubon, Maria Jose; Yu, Jinyeong; Choi, Sanghyuk; Park, Ki-Sook

2018-01-01

Transforming growth factor-beta (TGF-β) induces the migration and mobilization of bone marrow-derived mesenchymal stem cells (BM-MSCs) to maintain bone homeostasis during bone remodeling and facilitate the repair of peripheral tissues. Although many studies have reported the mechanisms through which TGF-β mediates the migration of various types of cells, including cancer cells, the intrinsic cellular mechanisms underlying cellular migration, and mobilization of BM-MSCs mediated by TGF-β are unclear. In this study, we showed that TGF-β activated noncanonical signaling molecules, such as Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), focal adhesion kinase (FAK), and p38, via TGF-β type I receptor in human BM-MSCs and murine BM-MSC-like ST2 cells. Inhibition of Rac1 by NSC23766 and Src by PP2 resulted in impaired TGF-β-mediated migration. These results suggested that the Smad-independent, noncanonical signals activated by TGF-β were necessary for migration. We also showed that N-cadherin-dependent intercellular interactions were required for TGF-β-mediated migration using functional inhibition of N-cadherin with EDTA treatment and a neutralizing antibody (GC-4 antibody) or siRNA-mediated knockdown of N-cadherin. However, N-cadherin knockdown did not affect the global activation of noncanonical signals in response to TGF-β. Therefore, these results suggested that the migration of BM-MSCs in response to TGF-β was mediated through N-cadherin and noncanonical TGF-β signals. © 2017 Wiley Periodicals, Inc.

Establishment of cell-cell junctions depends on the oligomeric states of VE-cadherin

PubMed Central

Bibert, Stéphanie; Ayari, Hélène; Riveline, Daniel; Concord, Evelyne; Hermant, Bastien; Vernet, Thierry; Gulino-Debrac, Danièle

2008-01-01

Specifically expressed at intercellular adherens junctions of endothelial cells, VE-cadherin is a receptor that exhibits particular self-association properties. Indeed, in vitro studies demonstrated that the extracellular part of VE-cadherin elaborates Ca++-dependent hexameric structures. We hypothesized that this assembly could be at the basis of a new cadherin-mediated cell-cell adhesion mechanism. To verify this assumption, we first demonstrated that VE-cadherin can elaborate hexamers at the cell surface of confluent endothelial cells. Second, mutations were introduced within the extracellular part of VE-cadherin to destabilize the hexamer. Following an in vitro screening, three mutants were selected, among which, one is able to elaborate only dimers. The selected mutations were expressed as C-terminal Green Fluorescent Protein fusions in CHO cells. Despite their capacity to elaborate nascent cell-cell contacts, the mutants seem to be rapidly degraded and or internalized. Altogether, our results suggest that the formation of VE-cadherin hexamers protects this receptor and might allow the elaboration of mature endothelial cell-cell junctions. PMID:18343874

Inter-Cellular Exchange of Cellular Components via VE-Cadherin-Dependent Trans-Endocytosis

PubMed Central

Sakurai, Takashi; Woolls, Melissa J.; Jin, Suk-Won

2014-01-01

Cell-cell communications typically involve receptor-mediated signaling initiated by soluble or cell-bound ligands. Here, we report a unique mode of endocytosis: proteins originating from cell-cell junctions and cytosolic cellular components from the neighboring cell are internalized, leading to direct exchange of cellular components between two adjacent endothelial cells. VE-cadherins form transcellular bridges between two endothelial cells that are the basis of adherence junctions. At such adherens junction sites, we observed the movement of the entire VE-cadherin molecule from one endothelial cell into the other with junctional and cytoplasmic components. This phenomenon, here termed trans-endocytosis, requires the establishment of a VE-cadherin homodimer in trans with internalization proceeding in a Rac1-, and actomyosin-dependent manner. Importantly, the trans-endocytosis is not dependent on any known endocytic pathway including clathrin-dependent endocytosis, macropinocytosis or phagocytosis. This novel form of cell-cell communications, leading to a direct exchange of cellular components, was observed in 2D and 3D-cultured endothelial cells as well as in the developing zebrafish vasculature. PMID:24603875

Transcriptional Elongation Regulator 1 Affects Transcription and Splicing of Genes Associated with Cellular Morphology and Cytoskeleton Dynamics and Is Required for Neurite Outgrowth in Neuroblastoma Cells and Primary Neuronal Cultures.

PubMed

Muñoz-Cobo, Juan Pablo; Sánchez-Hernández, Noemí; Gutiérrez, Sara; El Yousfi, Younes; Montes, Marta; Gallego, Carme; Hernández-Munain, Cristina; Suñé, Carlos

2017-12-01

TCERG1 is a highly conserved human protein implicated in interactions with the transcriptional and splicing machinery that is associated with neurodegenerative disorders. Biochemical, neuropathological, and genetic evidence suggests an important role for TCERG1 in Huntington's disease (HD) pathogenesis. At present, the molecular mechanism underlying TCERG1-mediated neuronal effects is unknown. Here, we show that TCERG1 depletion led to widespread alterations in mRNA processing that affected different types of alternative transcriptional or splicing events, indicating that TCERG1 plays a broad role in the regulation of alternative splicing. We observed considerable changes in the transcription and alternative splicing patterns of genes involved in cytoskeleton dynamics and neurite outgrowth. Accordingly, TCERG1 depletion in the neuroblastoma SH-SY5Y cell line and primary mouse neurons affected morphogenesis and resulted in reduced dendritic outgrowth, with a major effect on dendrite ramification and branching complexity. These defects could be rescued by ectopic expression of TCERG1. Our results indicate that TCERG1 affects expression of multiple mRNAs involved in neuron projection development, whose misregulation may be involved in TCERG1-linked neurological disorders.

Molecular determinants of cadherin ideal bond formation: Conformation-dependent unbinding on a multidimensional landscape

PubMed Central

Manibog, Kristine; Sankar, Kannan; Kim, Sun-Ae; Zhang, Yunxiang; Jernigan, Robert L.; Sivasankar, Sanjeevi

2016-01-01

Classical cadherin cell–cell adhesion proteins are essential for the formation and maintenance of tissue structures; their primary function is to physically couple neighboring cells and withstand mechanical force. Cadherins from opposing cells bind in two distinct trans conformations: strand-swap dimers and X-dimers. As cadherins convert between these conformations, they form ideal bonds (i.e., adhesive interactions that are insensitive to force). However, the biophysical mechanism for ideal bond formation is unknown. Here, we integrate single-molecule force measurements with coarse-grained and atomistic simulations to resolve the mechanistic basis for cadherin ideal bond formation. Using simulations, we predict the energy landscape for cadherin adhesion, the transition pathways for interconversion between X-dimers and strand-swap dimers, and the cadherin structures that form ideal bonds. Based on these predictions, we engineer cadherin mutants that promote or inhibit ideal bond formation and measure their force-dependent kinetics using single-molecule force-clamp measurements with an atomic force microscope. Our data establish that cadherins adopt an intermediate conformation as they shuttle between X-dimers and strand-swap dimers; pulling on this conformation induces a torsional motion perpendicular to the pulling direction that unbinds the proteins and forms force-independent ideal bonds. Torsional motion is blocked when cadherins associate laterally in a cis orientation, suggesting that ideal bonds may play a role in mechanically regulating cadherin clustering on cell surfaces. PMID:27621473

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.

Quantitative assessment of neural outgrowth using spatial light interference microscopy

NASA Astrophysics Data System (ADS)

Lee, Young Jae; Cintora, Pati; Arikkath, Jyothi; Akinsola, Olaoluwa; Kandel, Mikhail; Popescu, Gabriel; Best-Popescu, Catherine

2017-06-01

Optimal growth as well as branching of axons and dendrites is critical for the nervous system function. Neuritic length, arborization, and growth rate determine the innervation properties of neurons and define each cell's computational capability. Thus, to investigate the nervous system function, we need to develop methods and instrumentation techniques capable of quantifying various aspects of neural network formation: neuron process extension, retraction, stability, and branching. During the last three decades, fluorescence microscopy has yielded enormous advances in our understanding of neurobiology. While fluorescent markers provide valuable specificity to imaging, photobleaching, and photoxicity often limit the duration of the investigation. Here, we used spatial light interference microscopy (SLIM) to measure quantitatively neurite outgrowth as a function of cell confluence. Because it is label-free and nondestructive, SLIM allows for long-term investigation over many hours. We found that neurons exhibit a higher growth rate of neurite length in low-confluence versus medium- and high-confluence conditions. We believe this methodology will aid investigators in performing unbiased, nondestructive analysis of morphometric neuronal parameters.

Tuberin-deficiency downregulates N-cadherin and upregulates vimentin in kidney tumor of TSC patients

PubMed Central

Liang, Sitai; Salas, Tiffanie; Gencaslan, Emre; Li, Baojie; Habib, Samy L.

2014-01-01

Angiomyolipomas (AMLs) are associated with cell fibrosis in kidney of Tuberous Sclerosis Complex patients. The mechanism by which the fibrotic proteins accumulated in AMLs has not been explored. In the present study, we investigated the role of Akt/tuberin/mTOR pathway in the regulation cell fibrosis proteins. AML cells that expressed low levels of tuberin showed less expression of N-cadherin and higher of vimentin proteins compared to HEK293 cells. AML cells infected with Ad-tuberin showed a significant decrease in vimentin and an increase in N-cadherin protein expression. In addition, cells treated with rapamycin showed a significant increase in p-Akt and a decrease in p-p70S6K that was associated with a decrease expression of vimentin and a slight increase expression in N-cadherin. On the other hand, cells treated with Akt inhibitor revealed a significant decrease in p-Akt and p-p70S6K that was associated with a significant decrease in vimentin and an increase in N-cadherin expression. In addition, cells transfected with DN-Akt or DN-S6K show significant increase expression in N-cadherin and a decrease in vimentin. Moreover, cells transfected with siRNA against rictor or siRNA against raptor resulted in a decrease in vimentin and an increase N-cadherin expression. Kidney tumors from TSC patients showed significant decrease in N-cadherin and significant increased in vimentin protein expression compared to control kidney tissues. These data comprise the first report to provide the role of Akt/tuberin/mTORC1/2 in the regulation of N-cadherin and vimentin that are involved in the progression of fibrosis in kidney tumor of TSC patients. PMID:25149531

DNA topoisomerase IIβ stimulates neurite outgrowth in neural differentiated human mesenchymal stem cells through regulation of Rho-GTPases (RhoA/Rock2 pathway) and Nurr1 expression.

PubMed

Zaim, Merve; Isik, Sevim

2018-04-25

DNA topoisomerase IIβ (topo IIβ) is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance. However, the pathways of axon growth controlled by topo IIβ have not been clarified yet. Microarray results of our previous study have shown that topo IIβ silencing in neural differentiated primary human mesenchymal stem cells (hMSCs) significantly alters the expression pattern of genes involved in neural polarity, axonal growth, and guidance, including Rho-GTPases. This study aims to further analyze the regulatory role of topo IIβ on the process of axon growth via regulation of Rho-GTPases. For this purpose, topo IIβ was silenced in neurally differentiated hMSCs. Cells lost their morphology because of topo IIβ deficiency, becoming enlarged and flattened. Additionally, a reduction in both neural differentiation efficiency and neurite length, upregulation in RhoA and Rock2, downregulation in Cdc42 gene expression were detected. On the other hand, cells were transfected with topo IIβ gene to elucidate the possible neuroprotective effect of topo IIβ overexpression on neural-induced hMSCs. Topo IIβ overexpression prompted all the cells to exhibit neural cell morphology as characterized by longer neurites. RhoA and Rock2 expressions were downregulated, whereas Cdc42 expression was upregulated. Nurr1 expression level correlated with topo IIβ in both topo IIβ-overexpressed and -silenced cells. Furthermore, differential translocation of Rho-GTPases was detected by immunostaining in response to topo IIβ. Our results suggest that topo IIβ deficiency could give rise to neurodegeneration through dysregulation of Rho-GTPases. However, further in-vivo research is needed to demonstrate if re-regulation of Rho GTPases by topo IIβ overexpression could be a neuroprotective treatment in the case of neurodegenerative diseases.

Calcium response and FcepsilonRI expression in bone marrow-derived mast cells co-cultured with SCG neurites.

PubMed

Suzuki, Akio; Suzuki, Ryo; Furuno, Tadahide; Teshima, Reiko; Nakanishi, Mamoru

2005-10-01

Communication between nerves and mast cells is a prototypic demonstration of neuro-immune interaction. Numerous studies have shown that the stimulation of nerves (or addition of neurotransmitters) can evoke activation of mast cells, and that mast cell-derived mediators can influence neuronal activity. However, it is still unknown whether high affinity IgE receptors (FcepsilonRI) themselves are involved directly in the communication between nerves and mast cells. In the present experiments, we used an in vitro co-culture approach comprising interaction between immune (bone marrow-derived mast cells, BMMCs) and nerve cells (superior cervical ganglia, SCG) to solve the above problem. We found that the intracellular calcium ion concentration ([Ca2+]i) increased much more in BMMCs after antigen (DNP7-BSA) stimulation when they were associated with SCG neurites in the co-culture system. But the [Ca2+]i in BMMCs was less increased when they were not associated with the neurites. Further, the in vitro co-culture approach of BMMCs with SCG neurites for 3 d showed the increases of FcepsilonRI expression occurred on the plasma membranes of BMMCs which were attached to the neurites. On the contrary, N-cadherin molecules which localized on the interface between on the plasma membrane of BMMCs and SCG neurites did not increase with the co-culture for 3 d. All of these results indicated that co-culturing BMMCs with SCG neurites for 3 d promoted not only the calcium response but also the FcepsilonRI expression in BMMCs.

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K-AKT and ERK signaling pathways.

PubMed

Zhou, C; Li, C; Li, D; Wang, Y; Shao, W; You, Y; Peng, J; Zhang, X; Lu, L; Shen, X

2013-12-19

The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K-AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K-AKT and ERK signaling pathways which are critical in neurite development. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Human Langerhans cells express E-cadherin.

PubMed

Blauvelt, A; Katz, S I; Udey, M C

1995-02-01

Murine Langerhans cells (LC) synthesize and express E-cadherin, a Ca(++)-dependent homophilic cell adhesion molecule that mediates LC-keratinocyte (KC) binding in vitro. In vivo, E-cadherin expression by LC may promote localization and persistence of LC within the epidermis through LC-KC adhesion. In addition, changes in LC E-cadherin expression or affinity may be an important factor in the egress of LC from the epidermis after exposure to antigen. The aim of the present study was to determine if human LC also express E-cadherin. Suction blister roofs were obtained from normal volunteers and epidermal cell (EC) suspensions were prepared by limited trypsinization in the presence of 1 mM Ca++. EC were then incubated with antibodies to E-cadherin and CD1a or HLA-DR, and examined by two-color analytical flow cytometry or immunofluorescence microscopy. Most (82.9% +/- 7.4% [mean +/- SD], range 67-89%, n = 7) freshly prepared human LC expressed E-cadherin, as did the majority of KC. The amount of E-cadherin (as determined by mean fluorescence intensity) expressed by LC and KC was similar. Trypsin/EDTA treatment of freshly prepared EC abrogated expression of E-cadherin by LC and KC, whereas E-cadherin was not degraded by trypsin in the presence of Ca++. LC expressed lower levels of E-cadherin after 3 d in culture. Thus, human LC, like murine LC, express the homophilic adhesion molecule E-cadherin, which may be important in establishing and maintaining interactions between LC and KC in mammalian epidermis.

ProNGF promotes neurite growth from a subset of NGF-dependent neurons by a p75NTR-dependent mechanism

PubMed Central

Howard, Laura; Wyatt, Sean; Nagappan, Guhan; Davies, Alun M.

2013-01-01

The somatosensory and sympathetic innervation of the vertebrate head is derived principally from the neurons of trigeminal and superior cervical ganglia (SCG), respectively. During development, the survival of both populations of neurons and the terminal growth and branching of their axons in the tissues they innervate is regulated by the supply of nerve growth factor (NGF) produced by these tissues. NGF is derived by proteolytic cleavage of a large precursor protein, proNGF, which is recognised to possess distinctive biological functions. Here, we show that proNGF promotes profuse neurite growth and branching from cultured postnatal mouse SCG neurons. In marked contrast, proNGF does not promote the growth of trigeminal neurites. Studies using compartment cultures demonstrated that proNGF acts locally on SCG neurites to promote growth. The neurite growth-promoting effect of proNGF is not observed in SCG neurons cultured from p75NTR-deficient mice, and proNGF does not phosphorylate the NGF receptor tyrosine kinase TrkA. These findings suggest that proNGF selectively promotes the growth of neurites from a subset of NGF-responsive neurons by a p75NTR-dependent mechanism during postnatal development when the axons of these neurons are ramifying within their targets in vivo. PMID:23633509

Myelin-induced inhibition in a spiral ganglion organ culture - Approaching a natural environment in vitro.

PubMed

Kramer, Benedikt; Tropitzsch, Anke; Müller, Marcus; Löwenheim, Hubert

2017-08-15

The performance of a cochlear implant depends on the defined interaction between afferent neurons of the spiral ganglion and the inserted electrode. Neurite outgrowth can be induced by neurotrophins such as brain-derived neurotrophic factor (BDNF) via tropomyosin kinase receptor B (TrkB). However, neurotrophin signaling through the p75 neurotrophin receptor (p75) inhibits neurite outgrowth in the presence of myelin. Organotypic cultures derived from postnatal (P3-5) mice were used to study myelin-induced inhibition in the cochlear spiral ganglion. Neurite outgrowth was analyzed and quantified utilizing an adapted Sholl analysis. Stimulation of neurite outgrowth was quantified after application of BDNF, the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) and a selective inhibitor of the Rho-associated kinase (Y27632), which inhibits the p75 pathway. Myelin-induced inhibition was assessed by application of myelin-associated glycoprotein (MAG-Fc) to stimulate the inhibitory p75 pathway. Inhibition of neurite outgrowth was achieved by the selective TrkB inhibitor K252a. Stimulation of neurite outgrowth was observed after treatment with BDNF, 7,8 DHF and a combination of BDNF and Y27632. The 7,8-DHF-induced growth effects could be inhibited by K252a. Furthermore, inhibition of neurite outgrowth was observed after supplementation with MAG-Fc. Myelin-induced inhibition could be overcome by 7,8-DHF and the combination of BDNF and Y27632. In this study, myelin-induced inhibition of neurite outgrowth was established in a spiral ganglion model. We reveal that 7,8-DHF is a viable novel compound for the stimulation of neurite outgrowth in a myelin-induced inhibitory environment. The combination of TrkB stimulation and ROCK inhibition can be used to overcome myelin inhibition. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Sip1 mediates an E-cadherin-to-N-cadherin switch during cranial neural crest EMT

PubMed Central

Rogers, Crystal D.; Saxena, Ankur

2013-01-01

The neural crest, an embryonic stem cell population, initially resides within the dorsal neural tube but subsequently undergoes an epithelial-to-mesenchymal transition (EMT) to commence migration. Although neural crest and cancer EMTs are morphologically similar, little is known regarding conservation of their underlying molecular mechanisms. We report that Sip1, which is involved in cancer EMT, plays a critical role in promoting the neural crest cell transition to a mesenchymal state. Sip1 transcripts are expressed in premigratory/migrating crest cells. After Sip1 loss, the neural crest specifier gene FoxD3 was abnormally retained in the dorsal neuroepithelium, whereas Sox10, which is normally required for emigration, was diminished. Subsequently, clumps of adherent neural crest cells remained adjacent to the neural tube and aberrantly expressed E-cadherin while lacking N-cadherin. These findings demonstrate two distinct phases of neural crest EMT, detachment and mesenchymalization, with the latter involving a novel requirement for Sip1 in regulation of cadherin expression during completion of neural crest EMT. PMID:24297751

Magnolol Inhibits Human Glioblastoma Cell Migration by Regulating N-Cadherin.

PubMed

Cheng, Yu-Chen; Tsao, Min-Jen; Chiu, Chen-Yang; Kan, Po-Chieh; Chen, Ying

2018-06-01

Glioblastoma is a primary malignant brain tumor with a poor prognosis. An effective treatment for glioblastoma is needed. Magnolol is a natural compound from Magnolia officinalis suggested to have antiproliferative activity. The aim of this research was to investigate the anticancer effects of magnolol in glioma, with an emphasis on migration and the underlying mechanism. Magnolol decreased the expression of focal adhesion-related proteins and inhibited LN229 and U87MG glioma cell migration. The levels of phosphorylated myosin light chain (p-MLC), phosphorylated myosin light chain kinase and myosin phosphatase target subunit 1 were reduced in response to magnolol treatment. In addition, immunostaining and membrane fractionation showed that the distribution of N-cadherin at the glioma cell membrane was decreased by magnolol. In an orthotropic xenograft animal model, magnolol treatment not only inhibited tumor progression but also reduced p-MLC and N-cadherin protein expression. In conclusion, magnolol reduces cell migration, potentially through regulating focal adhesions and N-cadherin in glioma cells. Magnolol is a potential candidate for glioma treatment.

Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation.

PubMed

Chung, Heesung; Jung, Hyejung; Jho, Eek-Hoon; Multhaupt, Hinke A B; Couchman, John R; Oh, Eok-Soo

2018-06-14

In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, -3 and -6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation. Copyright © 2018. Published by Elsevier Inc.

Activation of RhoA by Lysophosphatidic Acid and Gα12/13 Subunits in Neuronal Cells: Induction of Neurite Retraction

PubMed Central

Kranenburg, Onno; Poland, Mieke; van Horck, Francis P. G.; Drechsel, David; Hall, Alan; Moolenaar, Wouter H.

1999-01-01

Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein–coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Gα12 and Gα13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity. PMID:10359601

Vitamin D regulates tyrosine hydroxylase expression: N-cadherin a possible mediator.

PubMed

Cui, X; Pertile, R; Liu, P; Eyles, D W

2015-09-24

Vitamin D is a neuroactive steroid. Its genomic actions are mediated via the active form of vitamin D, 1,25(OH)2D3, binding to the vitamin D receptor (VDR). The VDR emerges in the rat mesencephalon at embryonic day 12, representing the peak period of dopaminergic cell birth. Our prior studies reveal that developmental vitamin D (DVD)-deficiency alters the ontogeny of dopaminergic neurons in the developing mesencephalon. There is also consistent evidence from others that 1,25(OH)2D3 promotes the survival of dopaminergic neurons in models of dopaminergic toxicity. In both developmental and toxicological studies it has been proposed that 1,25(OH)2D3 may modulate the differentiation and maturation of dopaminergic neurons; however, to date there is lack of direct evidence. The aim of the current study is to investigate this both in vitro using a human SH-SY5Y cell line transfected with rodent VDR and in vivo using a DVD-deficient model. Here we show that in VDR-expressing SH-SY5Y cells, 1,25(OH)2D3 significantly increased production of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. This effect was dose- and time-dependent, but was not due to an increase in TH-positive cell number, nor was it due to the production of trophic survival factors for dopamine neurons such as glial-derived neurotrophic factor (GDNF). In accordance with 1,25(OH)2D3's anti-proliferative actions in the brain, 1,25(OH)2D3 reduced the percentage of dividing cells from approximately 15-10%. Given the recently reported role of N-cadherin in the direct differentiation of dopaminergic neurons, we examined here whether it may be elevated by 1,25(OH)2D3. We confirmed this in vitro and more importantly, we showed DVD-deficiency decreases N-cadherin expression in the embryonic mesencephalon. In summary, in our in vitro model we have shown 1,25(OH)2D3 increases TH expression, decreases proliferation and elevates N-cadherin, a potential factor that mediates these processes

N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes.

PubMed

Hertig, C M; Butz, S; Koch, S; Eppenberger-Eberhardt, M; Kemler, R; Eppenberger, H M

1996-01-01

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The 'redifferentiation model' of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the

Increased levels of fucosyltransferase IX and carbohydrate Lewis(x) adhesion determinant in human NT2N neurons.

PubMed

Brito, Catarina; Escrevente, Cristina; Reis, Celso A; Lee, Virginia M-Y; Trojanowski, John Q; Costa, Júlia

2007-05-01

The expression of the fucosylated carbohydrate Lewis(x) (Le(x)) determinant (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R) has been found in glycoproteins, proteoglycans, and glycolipids from the nervous system. Evidence suggests its association with cell-cell recognition, neurite outgrowth, and neuronal migration during central nervous system development. In the present work, we detected increased levels of Le(x) in differentiated human NT2N neurons cultured in vitro. To identify which fucosyltransferase (FUT) synthesized the Le(x) in NT2N neurons, RT-PCR, FUT substrate specificity and Western blot analysis were carried out. Strong activity toward acceptors Galbeta4GlcNAc-O-R and Fucalpha2Galbeta4GlcNAc-O-R [R = -(CH(2))(3)NHCO(CH(2))(5)NH-biotin], together with strong FUT9 detection by Western blot and presence of transcripts showed that FUT9 was the enzyme associated with Le(x) biosynthesis in NT2N neurons. Le(x) was detected at the plasma membrane of NT2N neurons, in lysosomes marked with lysosomal-associated membrane protein 1 (LAMP-1), and it was found for the first time to colocalize with the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) that defines the TI-VAMP exocytic compartment that is involved in neurite outgrowth. Furthermore, incubation with anti-Le(x) monoclonal antibody L5 led to impaired adhesion of NT2N neurons to the surface matrix and inhibited neurite initiation. In conclusion, FUT9 and its product Le(x) are detected specifically in human NT2N neurons and our results indicate that they underlie cell differentiation, cell adhesion, and initiation of neurite outgrowth in those neurons. (c) 2007 Wiley-Liss, Inc.

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

A natural diarylheptanoid promotes neuronal differentiation via activating ERK and PI3K-Akt dependent pathways.

PubMed

Tang, G; Dong, X; Huang, X; Huang, X-J; Liu, H; Wang, Y; Ye, W-C; Shi, L

2015-09-10

Neuronal differentiation is a critical developmental process that determines accurate synaptic connection and circuit wiring. A wide variety of naturally occurring compounds have been shown as promising drug leads for the generation and differentiation of neurons. Here we report that a diarylheptanoid from the plant Alpinia officinarum, 7-(4-hydroxyphenyl)-1-phenyl-4E-hepten-3-one (Cpd 1), exhibited potent activities in neuronal differentiation and neurite outgrowth. Cpd 1 induced differentiation of neuroblastoma Neuro-2a cells into a neuron-like morphology, and accelerated the establishment of axon-dendrite polarization of cultured hippocampal neurons. Moreover, Cpd 1 promoted neurite extension in both Neuro-2a cells and neurons. We showed that the effects of Cpd 1 on neuronal differentiation and neurite growth were specifically dependent on the activation of extracellular signal-regulated kinases (ERKs) and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways. Importantly, intraperitoneal administration of Cpd 1 promoted the differentiation of new-born progenitor cells into mature neurons in the adult hippocampal dentate gyrus. Collectively, this study identifies a naturally occurring diarylheptanoid with beneficial effects on neuronal differentiation and neurite outgrowth in vitro and in vivo. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

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

NHERF Links the N-Cadherin/Catenin Complex to the Platelet-derived Growth Factor Receptor to Modulate the Actin Cytoskeleton and Regulate Cell Motility

PubMed Central

Theisen, Christopher S.; Wahl, James K.; Johnson, Keith R.

2007-01-01

Using phage display, we identified Na+/H+ exchanger regulatory factor (NHERF)-2 as a novel binding partner for the cadherin-associated protein, β-catenin. We showed that the second of two PSD-95/Dlg/ZO-1 (PDZ) domains of NHERF interacts with a PDZ-binding motif at the very carboxy terminus of β-catenin. N-cadherin expression has been shown to induce motility in a number of cell types. The first PDZ domain of NHERF is known to bind platelet-derived growth factor-receptor β (PDGF-Rβ), and the interaction of PDGF-Rβ with NHERF leads to enhanced cell spreading and motility. Here we show that β-catenin and N-cadherin are in a complex with NHERF and PDGF-Rβ at membrane ruffles in the highly invasive fibrosarcoma cell line HT1080. Using a stable short hairpin RNA system, we showed that HT1080 cells knocked down for either N-cadherin or NHERF had impaired ability to migrate into the wounded area in a scratch assay, similar to cells treated with a PDGF-R kinase inhibitor. Cells expressing a mutant NHERF that is unable to associate with β-catenin had increased stress fibers, reduced lamellipodia, and impaired cell migration. Using HeLa cells, which express little to no PDGF-R, we introduced PDGF-Rβ and showed that it coimmunoprecipitates with N-cadherin and that PDGF-dependent cell migration was reduced in these cells when we knocked-down expression of N-cadherin or NHERF. These studies implicate N-cadherin and β-catenin in cell migration via PDGF-R–mediated signaling through the scaffolding molecule NHERF. PMID:17229887

Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer

PubMed Central

Carvalho, S; Catarino, TA; Dias, AM; Kato, M; Almeida, A; Hessling, B; Figueiredo, J; Gärtner, F; Sanches, JM; Ruppert, T; Miyoshi, E; Pierce, M; Carneiro, F; Kolarich, D; Seruca, R; Yamaguchi, Y; Taniguchi, N; Reis, CA; Pinho, SS

2016-01-01

E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell–cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression. PMID:26189796

Lithium alters the morphology of neurites regenerating from cultured adult spiral ganglion neurons.

PubMed

Shah, S M; Patel, C H; Feng, A S; Kollmar, R

2013-10-01

The small-molecule drug lithium (as a monovalent ion) promotes neurite regeneration and functional recovery, is easy to administer, and is approved for human use to treat bipolar disorder. Lithium exerts its neuritogenic effect mainly by inhibiting glycogen synthase kinase 3, a constitutively-active serine/threonine kinase that is regulated by neurotrophin and "wingless-related MMTV integration site" (Wnt) signaling. In spiral ganglion neurons of the cochlea, the effects of lithium and the function of glycogen synthase kinase 3 have not been investigated. We, therefore, set out to test whether lithium modulates neuritogenesis from adult spiral ganglion neurons. Primary cultures of dissociated spiral ganglion neurons from adult mice were exposed to lithium at concentrations between 0 and 12.5 mM. The resulting neurite morphology and growth-cone appearance were measured in detail by using immunofluorescence microscopy and image analysis. We found that lithium altered the morphology of regenerating neurites and their growth cones in a differential, concentration-dependent fashion. Low concentrations of 0.5-2.5 mM (around the half-maximal inhibitory concentration for glycogen synthase kinase 3 and the recommended therapeutic serum concentration for bipolar disorder) enhanced neurite sprouting and branching. A high concentration of 12.5 mM, in contrast, slowed elongation. As the lithium concentration rose from low to high, the microtubules became increasingly disarranged and the growth cones more arborized. Our results demonstrate that lithium selectively stimulates phases of neuritogenesis that are driven by microtubule reorganization. In contrast, most other drugs that have previously been tested on spiral ganglion neurons are reported to inhibit neurite outgrowth or affect only elongation. Lithium sensitivity is a necessary, but not sufficient condition for the involvement of glycogen synthase kinase 3. Our results are, therefore, consistent with, but do not prove

O-mannosylation and N-glycosylation: two coordinated mechanisms regulating the tumour suppressor functions of E-cadherin in cancer

PubMed Central

Bartels, Markus F.; Miyoshi, Eiji; Pierce, Michael; Taniguchi, Naoyuki; Carneiro, Fátima; Seruca, Raquel; Reis, Celso A.; Strahl, Sabine; Pinho, Salomé S.

2016-01-01

Dysregulation of tumor suppressor protein E-cadherin is an early molecular event in cancer. O-mannosylation profile of E-cadherin is a newly-described post-translational modification crucial for its adhesive functions in homeostasis. However, the role of O-mannosyl glycans in E-cadherin-mediated cell adhesion in cancer and their interplay with N-glycans remains largely unknown. We herein demonstrated that human gastric carcinomas exhibiting a non-functional E-cadherin display a reduced expression of O-mannosyl glycans concomitantly with increased modification with branched complex N-glycans. Accordingly, overexpression of MGAT5-mediated branched N-glycans both in gastric cancer cells and transgenic mice models led to a significant decrease of O-mannosyl glycans attached to E-cadherin that was associated with impairment of its tumour suppressive functions. Importantly, overexpression of protein O-mannosyltransferase 2 (POMT2) induced a reduced expression of branched N-glycans which led to a protective effect of E-cadherin biological functions. Overall, our results reveal a newly identified mechanism of (dys)regulation of E-cadherin that occur through the interplay between O-mannosylation and N-glycosylation pathway. PMID:27533452

Dose-Dependent Differential Effect of Neurotrophic Factors on In Vitro and In Vivo Regeneration of Motor and Sensory Neurons

PubMed Central

Santos, Daniel; Gonzalez-Perez, Francisco; Navarro, Xavier

2016-01-01

Although peripheral axons can regenerate after nerve transection and repair, functional recovery is usually poor due to inaccurate reinnervation. Neurotrophic factors promote directional guidance to regenerating axons and their selective application may help to improve functional recovery. Hence, we have characterized in organotypic cultures of spinal cord and dorsal root ganglia the effect of GDNF, FGF-2, NGF, NT-3, and BDNF at different concentrations on motor and sensory neurite outgrowth. In vitro results show that GDNF and FGF-2 enhanced both motor and sensory neurite outgrowth, NGF and NT-3 were the most selective to enhance sensory neurite outgrowth, and high doses of BDNF selectively enhanced motor neurite outgrowth. Then, NGF, NT-3, and BDNF (as the most selective factors) were delivered in a collagen matrix within a silicone tube to repair the severed sciatic nerve of rats. Quantification of Fluorogold retrolabeled neurons showed that NGF and NT-3 did not show preferential effect on sensory regeneration whereas BDNF preferentially promoted motor axons regeneration. Therefore, the selective effects of NGF and NT-3 shown in vitro are lost when they are applied in vivo, but a high dose of BDNF is able to selectively enhance motor neuron regeneration both in vitro and in vivo. PMID:27867665

Dendrite and Axon Specific Geometrical Transformation in Neurite Development

PubMed Central

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

2016-01-01

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

Cross-link regulation of precursor N-cadherin and FGFR1 by GDNF increases U251MG cell viability.

PubMed

Tang, Chuan-Xi; Gu, Yan-Xia; Liu, Xin-Feng; Tong, Shu-Yan; Ayanlaja, Abiola A; Gao, Yue; Ji, Guang-Quan; Xiong, Ye; Huang, Lin-Yan; Gao, Dian-Shuai

2018-07-01

Glial cell line-derived neurotrophic factor (GDNF) is considered to be involved in the development of glioma. However, uncovering the underlying mechanism of the proliferation of glioma cells is a challenging work in progress. We have identified the binding of the precursor of N-cadherin (proN-cadherin) and GDNF on the cell membrane in previous studies. In the present study, we observed increased U251 Malignant glioma (U251MG) cell viability by exogenous GDNF (50 ng/ml). We also confirmed that the high expression of the proN-cadherin was stimulated by exogenous GDNF. Concurrently, we affirmed that lower expression of proN-cadherin correlated with reduced glioma cell viability. Additionally, we observed glioma cell U251MG viability as the phosphorylation level of FGFR1 at Y653 and Y654 was increased after exogenous GDNF treatment, which led to increased interaction between proN-cadherin and FGFR1 (pY653+Y654). Our experiments presented a new mechanism adopted by GDNF supporting glioma development and indicated a possible therapeutic potential via the inhibition of proN-cadherin/FGFR1 interaction.

Growing Neural PC-12 Cell on Crosslinked Silica Aerogels Increases Neurite Extension in the Presence of an Electric Field.

PubMed

Lynch, Kyle J; Skalli, Omar; Sabri, Firouzeh

2018-04-20

Externally applied electrical stimulation (ES) has been shown to enhance the nerve regeneration process and to influence the directionality of neurite outgrowth. In addition, the physical and chemical properties of the substrate used for nerve-cell regeneration is critical in fostering regeneration. Previously, we have shown that polyurea-crosslinked silica aerogels (PCSA) exert a positive influence on the extension of neurites by PC-12 cells, a cell-line model widely used to study neurite extension and electrical excitability. In this work, we have examined how an externally applied electric field (EF) influences the extension of neurites in PC-12 cells grown on two substrates: collagen-coated dishes versus collagen-coated crosslinked silica aerogels. The externally applied direct current (DC) bias was applied in vitro using a custom-designed chamber containing polydimethysiloxane (PDMS) embedded copper electrodes to create an electric field across the substrate for the cultured PC-12 cells. Results suggest orientation preference towards the anode, and, on average, longer neurites in the presence of the applied DC bias than with 0 V DC bias. In addition, neurite length was increased in cells grown on silica-crosslinked aerogel when compared to cells grown on regular petri-dishes. These results further support the notion that PCSA is a promising material for nerve regeneration.

Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines.

PubMed

Sandquist, Elizabeth J; Somji, Seema; Dunlevy, Jane R; Garrett, Scott H; Zhou, Xu Dong; Slusser-Nore, Andrea; Sens, Donald A

2016-01-01

Epithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown. Real-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice. This study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin. The finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth. Mesenchymal to epithelial transition

Surface Expression of Precursor N-cadherin Promotes Tumor Cell Invasion12

PubMed Central

Maret, Deborah; Gruzglin, Eugenia; Sadr, Mohamad Seyed; Siu, Vincent; Shan, Weisong; Koch, Alexander W; Seidah, Nabil G; Del Maestro, Rolando F; Colman, David R

2010-01-01

The expression of N-cadherin (NCAD) has been shown to correlate with increased tumor cell motility and metastasis. However, NCAD-mediated adhesion is a robust phenomenon and therefore seems to be inconsistent with the “release” from intercellular adhesion required for invasion. We show that in the most invasive melanoma and brain tumor cells, altered posttranslational processing results in abundant nonadhesive precursor N-cadherin (proNCAD) at the cell surface, although total NCAD levels remain constant. We demonstrate that aberrantly processed proNCAD promotes cell migration and invasion in vitro. Furthermore, in human tumor specimens, we find high levels of proNCAD as well, supporting an overall conclusion that proNCAD and mature NCAD coexist on these tumor cell surfaces and that it is the ratio between these functionally antagonistic moieties that directly correlates with invasion potential. Our work provides insight into what may be a widespread mechanism for invasion and metastasis and challenges the current dogma of the functional roles played by classic cadherins in tumor progression. PMID:21170270

Mitochondria-targeted antioxidants protect against amyloid-beta toxicity in Alzheimer's disease neurons.

PubMed

Manczak, Maria; Mao, Peizhong; Calkins, Marcus J; Cornea, Anda; Reddy, Arubala P; Murphy, Michael P; Szeto, Hazel H; Park, Byung; Reddy, P Hemachandra

2010-01-01

The purpose of our study was to investigate the effects of the mitochondria-targeted antioxidants, MitoQ and SS31, and the anti-aging agent resveratrol on neurons from a mouse model (Tg2576 line) of Alzheimer's disease (AD) and on mouse neuroblastoma (N2a) cells incubated with the amyloid-beta (Abeta) peptide. Using electron and confocal microscopy, gene expression analysis, and biochemical methods, we studied mitochondrial structure and function and neurite outgrowth in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta. In N2a cells only incubated with the Abeta, we found increased expressions of mitochondrial fission genes and decreased expression of fusion genes and also decreased expression of peroxiredoxins. Electron microscopy of the N2a cells incubated with Abeta revealed a significantly increased number of mitochondria, indicating that Abeta fragments mitochondria. Biochemical analysis revealed that function is defective in mitochondria. Neurite outgrowth was significantly decreased in Abeta-incubated N2a cells, indicating that Abeta affects neurite outgrowth. However, in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta, abnormal expression of peroxiredoxins and mitochondrial structural genes were prevented and mitochondrial function was normal; intact mitochondria were present and neurite outgrowth was significantly increased. In primary neurons from amyloid-beta precursor protein transgenic mice that were treated with MitoQ and SS31, neurite outgrowth was significantly increased and cyclophilin D expression was significantly decreased. These findings suggest that MitoQ and SS31 prevent Abeta toxicity, which would warrant the study of MitoQ and SS31 as potential drugs to treat patients with AD.

Orexin A Inhibits Propofol-Induced Neurite Retraction by a Phospholipase D/Protein Kinase Cε-Dependent Mechanism in Neurons

PubMed Central

Björnström, Karin; Turina, Dean; Strid, Tobias; Sundqvist, Tommy; Eintrei, Christina

2014-01-01

Background The intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction. Methods In primary cortical cell cultures from newborn rats’ brains, live cell light microscopy was used to measure neurite retraction after propofol (2 µM) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKCε translocation inhibitor peptide. Changes in PKCε Ser729 phosphorylation were detected with Western blot. Results The neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKCε translocation inhibitor peptide. OA increases via PLD and propofol decreases PKCε Ser729 phosphorylation, a crucial step in the activation of PKCε. Conclusions Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKCε-mediated pathway, and PKCε maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge. PMID:24828410

N-Cadherin and Fibroblast Growth Factor Receptors crosstalk in the control of developmental and cancer cell migrations.

PubMed

Nguyen, Thao; Mège, René Marc

2016-11-01

Cell migrations are diverse. They constitutemajor morphogenetic driving forces during embryogenesis, but they contribute also to the loss of tissue homeostasis and cancer growth. Capabilities of cells to migrate as single cells or as collectives are controlled by internal and external signalling, leading to the reorganisation of their cytoskeleton as well as by the rebalancing of cell-matrix and cell-cell adhesions. Among the genes altered in numerous cancers, cadherins and growth factor receptors are of particular interest for cell migration regulation. In particular, cadherins such as N-cadherin and a class of growth factor receptors, namely FGFRs cooperate to regulate embryonic and cancer cell behaviours. In this review, we discuss on reciprocal crosstalk between N-cadherin and FGFRs during cell migration. Finally, we aim at clarifying the synergy between N-cadherin and FGFR signalling that ensure cellular reorganization during cell movements, mainly during cancer cell migration and metastasis but also during developmental processes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Heterogeneous Cadherin Expression and Multicellular Aggregate Dynamics in Ovarian Cancer Dissemination.

PubMed

Klymenko, Yuliya; Johnson, Jeffrey; Bos, Brandi; Lombard, Rachel; Campbell, Leigh; Loughran, Elizabeth; Stack, M Sharon

2017-07-01

Epithelial ovarian carcinoma spreads via shedding of cells and multicellular aggregates (MCAs) from the primary tumor into peritoneal cavity, with subsequent intraperitoneal tumor cell:mesothelial cell adhesion as a key early event in metastatic seeding. Evaluation of human tumor extracts and tissues confirms that well-differentiated ovarian tumors express abundant E-cadherin (Ecad), whereas advanced lesions exhibit upregulated N-cadherin (Ncad). Two expression patterns are observed: "mixed cadherin," in which distinct cells within the same tumor express either E- or Ncad, and "hybrid cadherin," wherein single tumor cell(s) simultaneously expresses both cadherins. We demonstrate striking cadherin-dependent differences in cell-cell interactions, MCA formation, and aggregate ultrastructure. Mesenchymal-type Ncad+ cells formed stable, highly cohesive solid spheroids, whereas Ecad+ epithelial-type cells generated loosely adhesive cell clusters covered by uniform microvilli. Generation of "mixed cadherin" MCAs using fluorescently tagged cell populations revealed preferential sorting into cadherin-dependent clusters, whereas mixing of cell lines with common cadherin profiles generated homogeneous aggregates. Recapitulation of the "hybrid cadherin" Ecad+/Ncad+ phenotype, via insertion of the CDH2 gene into Ecad+ cells, resulted in the ability to form heterogeneous clusters with Ncad+ cells, significantly enhanced adhesion to organotypic mesomimetic cultures and peritoneal explants, and increased both migration and matrix invasion. Alternatively, insertion of CDH1 gene into Ncad+ cells greatly reduced cell-to-collagen, cell-to-mesothelium, and cell-to-peritoneum adhesion. Acquisition of the hybrid cadherin phenotype resulted in altered MCA surface morphology with increased surface projections and increased cell proliferation. Overall, these findings support the hypothesis that MCA cadherin composition impacts intraperitoneal cell and MCA dynamics and thereby affects

Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines

PubMed Central

Sandquist, Elizabeth J.; Somji, Seema; Dunlevy, Jane R.; Garrett, Scott H.; Zhou, Xu Dong; Slusser-Nore, Andrea

2016-01-01

Background Epithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown. Methods Real-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice. Results This study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin. Conclusions The finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth

Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology

PubMed Central

Gebbink, Martijn F.B.G.; Kranenburg, Onno; Poland, Mieke; van Horck, Francis P.G.; Houssa, Brahim; Moolenaar, Wouter H.

1997-01-01

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth. PMID:9199174

Toward the Development of an Artificial Brain on a Micropatterned and Material-Regulated Biochip by Guiding and Promoting the Differentiation and Neurite Outgrowth of Neural Stem/Progenitor Cells.

PubMed

Liu, Yung-Chiang; Lee, I-Chi; Lei, Kin Fong

2018-02-14

An in vitro model mimicking the in vivo environment of the brain must be developed to study neural communication and regeneration and to obtain an understanding of cellular and molecular responses. In this work, a multilayered neural network was successfully constructed on a biochip by guiding and promoting neural stem/progenitor cell differentiation and network formation. The biochip consisted of 3 × 3 arrays of cultured wells connected with channels. Neurospheroids were cultured on polyelectrolyte multilayer (PEM) films in the culture wells. Neurite outgrowth and neural differentiation were guided and promoted by the micropatterns and the PEM films. After 5 days in culture, a 3 × 3 neural network was constructed on the biochip. The function and the connections of the network were evaluated by immunocytochemistry and impedance measurements. Neurons were generated and produced functional and recyclable synaptic vesicles. Moreover, the electrical connections of the neural network were confirmed by measuring the impedance across the neurospheroids. The current work facilitates the development of an artificial brain on a chip for investigations of electrical stimulations and recordings of multilayered neural communication and regeneration.

Focal adhesion kinase regulates neuronal growth, synaptic plasticity and hippocampus-dependent spatial learning and memory.

PubMed

Monje, Francisco J; Kim, Eun-Jung; Pollak, Daniela D; Cabatic, Maureen; Li, Lin; Baston, Arthur; Lubec, Gert

2012-01-01

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase abundantly expressed in the mammalian brain and highly enriched in neuronal growth cones. Inhibitory and facilitatory activities of FAK on neuronal growth have been reported and its role in neuritic outgrowth remains controversial. Unlike other tyrosine kinases, such as the neurotrophin receptors regulating neuronal growth and plasticity, the relevance of FAK for learning and memory in vivo has not been clearly defined yet. A comprehensive study aimed at determining the role of FAK in neuronal growth, neurotransmitter release and synaptic plasticity in hippocampal neurons and in hippocampus-dependent learning and memory was therefore undertaken using the mouse model. Gain- and loss-of-function experiments indicated that FAK is a critical regulator of hippocampal cell morphology. FAK mediated neurotrophin-induced neuritic outgrowth and FAK inhibition affected both miniature excitatory postsynaptic potentials and activity-dependent hippocampal long-term potentiation prompting us to explore the possible role of FAK in spatial learning and memory in vivo. Our data indicate that FAK has a growth-promoting effect, is importantly involved in the regulation of the synaptic function and mediates in vivo hippocampus-dependent spatial learning and memory. Copyright © 2011 S. Karger AG, Basel.

G Protein-regulated inducer of neurite outgrowth (GRIN) modulates Sprouty protein repression of mitogen-activated protein kinase (MAPK) activation by growth factor stimulation.

PubMed

Hwangpo, Tracy Anh; Jordan, J Dedrick; Premsrirut, Prem K; Jayamaran, Gomathi; Licht, Jonathan D; Iyengar, Ravi; Neves, Susana R

2012-04-20

Gα(o/i) interacts directly with GRIN (G protein-regulated inducer of neurite outgrowth). Using the yeast two-hybrid system, we identified Sprouty2 as an interacting partner of GRIN. Gα(o) and Sprouty2 bind to overlapping regions of GRIN, thus competing for GRIN binding. Imaging experiments demonstrated that Gα(o) expression promoted GRIN translocation to the plasma membrane, whereas Sprouty2 expression failed to do so. Given the role of Sprouty2 in the regulation of growth factor-mediated MAPK activation, we examined the contribution of the GRIN-Sprouty2 interaction to CB1 cannabinoid receptor regulation of FGF receptor signaling. In Neuro-2A cells, a system that expresses all of the components endogenously, modulation of GRIN levels led to regulation of MAPK activation. Overexpression of GRIN potentiated FGF activation of MAPK and decreased tyrosine phosphorylation of Sprouty2. Pretreatment with G(o/i)-coupled CB1 receptor agonist attenuated subsequent FGF activation of MAPK. Decreased expression of GRIN both diminished FGF activation of MAPK and blocked CB1R attenuation of MAPK activation. These observations indicate that Gα(o) interacts with GRIN and outcompetes GRIN from bound Sprouty. Free Sprouty then in turn inhibits growth factor signaling. Thus, here we present a novel mechanism of how G(o/i)-coupled receptors can inhibit growth factor signaling to MAPK.

Impact of pH on the structure and function of neural cadherin.

PubMed

Jungles, Jared M; Dukes, Matthew P; Vunnam, Nagamani; Pedigo, Susan

2014-12-02

Neural (N-) cadherin is a transmembrane protein within adherens junctions that mediates cell-cell adhesion. It has 5 modular extracellular domains (EC1-EC5) that bind 3 calcium ions between each of the modules. Calcium binding is required for dimerization. N-Cadherin is involved in diverse processes including tissue morphogenesis, excitatory synapse formation and dynamics, and metastasis of cancer. During neurotransmission and tumorigenesis, fluctuations in extracellular pH occur, causing tissue acidosis with associated physiological consequences. Studies reported here aim to determine the effect of pH on the dimerization properties of a truncated construct of N-cadherin containing EC1-EC2. Since N-cadherin is an anionic protein, we hypothesized that acidification of solution would cause an increase in stability of the apo protein, a decrease in the calcium-binding affinity, and a concomitant decrease in the formation of adhesive dimer. The stability of the apo monomer was increased and the calcium-binding affinity was decreased at reduced pH, consistent with our hypothesis. Surprisingly, analytical SEC studies showed an increase in calcium-induced dimerization as solution pH decreased from 7.4 to 5.0. Salt-dependent dimerization studies indicated that electrostatic repulsion attenuates dimerization affinity. These results point to a possible electrostatic mechanism for moderating dimerization affinity of the Type I cadherin family. Extrapolating these results to cell adhesion in vivo leads to the assertion that decreased pH promotes adhesion by N-cadherin, thereby stabilizing synaptic junctions.

The neurite growth inhibitory effects of soluble TNFα on developing sympathetic neurons are dependent on developmental age.

PubMed

Nolan, Aoife M; Collins, Louise M; Wyatt, Sean L; Gutierrez, Humberto; O'Keeffe, Gerard W

2014-01-01

During development, the growth of neural processes is regulated by an array of cellular and molecular mechanisms which influence growth rate, direction and branching. Recently, many members of the TNF superfamily have been shown to be key regulators of neurite growth during development. The founder member of this family, TNFα can both promote and inhibit neurite growth depending on the cellular context. Specifically, transmembrane TNFα promotes neurite growth, while soluble TNFα inhibits it. While the growth promoting effects of TNFα are restricted to a defined developmental window of early postnatal development, whether the growth inhibitory effects of soluble TNFα occur throughout development is unknown. In this study we used the extensively studied, well characterised neurons of the superior cervical ganglion to show that the growth inhibitory effects of soluble TNFα are restricted to a specific period of late embryonic and early postnatal development. Furthermore, we show that this growth inhibitory effect of soluble TNFα requires NF-κB signalling at all developmental stages at which soluble TNFα inhibits neurite growth. These findings raise the possibility that increases in the amount of soluble TNFα in vivo, for example as a result of maternal inflammation, could negatively affect neurite growth in developing neurons at specific stages of development. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues

PubMed Central

1989-01-01

P-cadherin is a subclass of Ca2+-dependent cell-cell adhesion molecules present in mouse placenta, where its localization suggests a function of connecting the embryo to the uterus (Nose, A., and M. Takeichi. 1986. J. Cell Biol. 103:2649-2658). We recently identified a human cadherin detected by an mAb capable of disrupting cell-cell adhesion of A-431 cells, and found that it was closely related immunochemically to mouse P-cadherin. Curiously, this cadherin was undetectable in human placenta by immunohistochemical examination (Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, and O. Abe. 1989. Cancer Res. 49:2128-2133). We here report the cloning and sequencing of cDNA clone encoding the human homologue of mouse P- cadherin. The deduced amino acid sequence of the human P-cadherin consists of 829 amino acid and shows striking homology with mouse P- cadherin. On Northern blot analysis, human P-cadherin was scarcely expressed in human placenta in contrast to mouse P-cadherin, which was abundantly expressed in mouse placenta throughout pregnancy, and it was shown that E-cadherin, but not P-cadherin, was the major cadherin molecule in human placenta. Moreover, NIH3T3 cells transfected with human P-cadherin cDNA expressed the functional cadherin molecule, which was identical to the cadherin we had previously identified using the mAb, showing that this molecule really does mediate cell-cell adhesion and that the cadherin we detected immunochemically is undoubtedly human P-cadherin. The results obtained in this study support the idea that P- cadherin plays little role, if any, in Ca2+-dependent cell-cell binding in human placental tissue at least after several weeks of pregnancy. PMID:2793940

Thrombomodulin reduces tumorigenic and metastatic potential of lung cancer cells by up-regulation of E-cadherin and down-regulation of N-cadherin expression.

PubMed

Zheng, Nana; Huo, Zihe; Zhang, Bin; Meng, Mei; Cao, Zhifei; Wang, Zhiwei; Zhou, Quansheng

2016-08-05

Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a "vascular protection god". In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery. Copyright © 2016 Elsevier Inc. All rights reserved.

Loss of function of KIAA2022 causes mild to severe intellectual disability with an autism spectrum disorder and impairs neurite outgrowth.

PubMed

Van Maldergem, Lionel; Hou, Qingming; Kalscheuer, Vera M; Rio, Marlène; Doco-Fenzy, Martine; Medeira, Ana; de Brouwer, Arjan P M; Cabrol, Christelle; Haas, Stefan A; Cacciagli, Pierre; Moutton, Sébastien; Landais, Emilie; Motte, Jacques; Colleaux, Laurence; Bonnet, Céline; Villard, Laurent; Dupont, Juliette; Man, Heng-Ye

2013-08-15

Existence of a discrete new X-linked intellectual disability (XLID) syndrome due to KIAA2022 deficiency was questioned by disruption of KIAA2022 by an X-chromosome pericentric inversion in a XLID family we reported in 2004. Three additional families with likely pathogenic KIAA2022 mutations were discovered within the frame of systematic parallel sequencing of familial cases of XLID or in the context of routine array-CGH evaluation of sporadic intellectual deficiency (ID) cases. The c.186delC and c.3597dupA KIAA2022 truncating mutations were identified by X-chromosome exome sequencing, while array CGH discovered a 70 kb microduplication encompassing KIAA2022 exon 1 in the third family. This duplication decreased KIAA2022 mRNA level in patients' lymphocytes by 60%. Detailed clinical examination of all patients, including the two initially reported, indicated moderate-to-severe ID with autistic features, strabismus in all patients, with no specific dysmorphic features other than a round face in infancy and no structural brain abnormalities on magnetic resonance imaging (MRI). Interestingly, the patient with decreased KIAA2022 expression had only mild ID with severe language delay and repetitive behaviors falling in the range of an autism spectrum disorder (ASD). Since little is known about KIAA2022 function, we conducted morphometric studies in cultured rat hippocampal neurons. We found that siRNA-mediated KIAA2022 knockdown resulted in marked impairment in neurite outgrowth including both the dendrites and the axons, suggesting a major role for KIAA2022 in neuron development and brain function.

E-Cadherin-Dependent Stimulation of Traction Force at Focal Adhesions via the Src and PI3K Signaling Pathways

PubMed Central

Jasaitis, Audrius; Estevez, Maruxa; Heysch, Julie; Ladoux, Benoit; Dufour, Sylvie

2012-01-01

The interplay between cadherin- and integrin-dependent signals controls cell behavior, but the precise mechanisms that regulate the strength of adhesion to the extracellular matrix remains poorly understood. We deposited cells expressing a defined repertoire of cadherins and integrins on fibronectin (FN)-coated polyacrylamide gels (FN-PAG) and on FN-coated pillars used as a micro-force sensor array (μFSA), and analyzed the functional relationship between these adhesion receptors to determine how it regulates cell traction force. We found that cadherin-mediated adhesion stimulated cell spreading on FN-PAG, and this was modulated by the substrate stiffness. We compared S180 cells with cells stably expressing different cadherins on μFSA and found that traction forces were stronger in cells expressing cadherins than in parental cells. E-cadherin-mediated contact and mechanical coupling between cells are required for this increase in cell-FN traction force, which was not observed in isolated cells, and required Src and PI3K activities. Traction forces were stronger in cells expressing type I cadherins than in cells expressing type II cadherins, which correlates with our previous observation of a higher intercellular adhesion strength developed by type I compared with type II cadherins. Our results reveal one of the mechanisms whereby molecular cross talk between cadherins and integrins upregulates traction forces at cell-FN adhesion sites, and thus provide additional insight into the molecular control of cell behavior. PMID:22853894

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

Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain

PubMed Central

Roehm, Pamela C.; Xu, Ningyong; Woodson, Erika A.; Green, Steven H.; Hansen, Marlan R.

2008-01-01

The effect of membrane electrical activity on spiral ganglion neuron (SGN) neurite growth remains unknown despite its relevance to cochlear implant technology. We demonstrate that membrane depolarization delays the initial formation and inhibits the subsequent extension of cultured SGN neurites. This inhibition depends directly on the level of depolarization with higher levels of depolarization causing retraction of existing neurites. Cultured SGNs express subunits for L-type, N-type, and P/Q type voltage-gated calcium channels (VGCCs) and removal of extracellular Ca2+ or treatment with a combination of L-type, N-type, P/Q-type VGCC antagonists rescues SGN neurite growth under depolarizing conditions. By measuring the fluorescence intensity of SGNs loaded with the fluorogenic calpain substrate t-butoxy carbonyl-Leu-Met-chloromethylaminocoumarin (20 μM), we demonstrate that depolarization activates calpains. Calpeptin (15 μM), a calpain inhibitor, prevents calpain activation by depolarization and rescues neurite growth in depolarized SGNs suggesting that calpain activation contributes to the inhibition of neurite growth by depolarization. PMID:18055215

Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition.

PubMed

Li, Qisheng; Sodroski, Catherine; Lowey, Brianna; Schweitzer, Cameron J; Cha, Helen; Zhang, Fang; Liang, T Jake

2016-07-05

Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

Cytoplasmic p21(Cip1/WAF1) regulates neurite remodeling by inhibiting Rho-kinase activity.

PubMed

Tanaka, Hiroyuki; Yamashita, Toshihide; Asada, Minoru; Mizutani, Shuki; Yoshikawa, Hideki; Tohyama, Masaya

2002-07-22

p21(Cip1/WAF1) has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21(Cip1/WAF1) is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21(Cip1/WAF1) lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21(Cip1/WAF1) is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21(Cip1/WAF1) may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions.

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

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

PubMed Central

Chazeau, Anaël; Garcia, Mikael; Czöndör, Katalin; Perrais, David; Tessier, Béatrice; Giannone, Grégory; Thoumine, Olivier

2015-01-01

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin–coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain. PMID:25568337

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

Complex interactions amongst N-cadherin, DLAR, and Liprin-α regulate Drosophila photoreceptor axon targeting

PubMed Central

Prakash, Saurabh; Maclendon, Helen; Dubreuil, Catherine I.; Ghose, Aurnab; Hwa, Jennifer; Dennehy, Kelly A.; Tomalty, Katharine M.H.; Clark, Kelsey; Van Vactor, David; Clandinin, Thomas R.

2009-01-01

The formation of stable adhesive contacts between pre- and post-synaptic neurons represents the initial step in synapse assembly. The cell adhesion molecule N-cadherin, the receptor tyrosine phosphatase DLAR, and the scaffolding molecule Liprin-α play critical, evolutionarily conserved roles in this process. However, how these proteins signal to the growth cone, and are themselves regulated, remains poorly understood. Using Drosophila photoreceptors (R cells) as a model, we evaluate genetic and physical interactions among these three proteins. We demonstrate that DLAR function in this context is independent of phosphatase activity, but requires interactions mediated by its intracellular domain. Genetic studies reveal both positive and, surprisingly, inhibitory interactions amongst all three genes. These observations are corroborated by biochemical studies demonstrating that DLAR physically associates via its phosphatase domain with N-cadherin in Drosophila embryos. Together, these data demonstrate that N-cadherin, DLAR, and Liprin-α function in a complex to regulate adhesive interactions between pre- and post-synaptic cells, and provide a novel mechanism for controlling the activity of liprin-α in the developing growth cone. PMID:19766621

Hypoxia reduces the E-cadherin expression and increases OSCC cell migration regardless of the E-cadherin methylation profile.

PubMed

Domingos, Patrícia Luciana Batista; Souza, Marcela Gonçalves; Guimarães, Talita Antunes; Santos, Eliane Sobrinho; Farias, Lucyana Conceição; de Carvalho Fraga, Carlos Alberto; Jones, Kimberly Marie; Santos, Sérgio Henrique Souza; de Paula, Alfredo Maurício Batista; Guimarães, André Luiz Sena

2017-05-01

The purpose of the current study is to investigate the association between E-cadherin methylation status, hypoxia and OSCC. HaCat and SCC9 cell lines were submitted to hypoxic treatment, followed by methylation profile analysis (MS-PCR) and analysis of the expression of mRNA gene E-cadherin (RT-PCR). Study group samples comprise individuals affected by potentially malignant lesions Potential Malignant Oral Lesion (PMOL, n=18) and oral squamous cell carcinoma (OSCC, n=28). The control group oral mucosa (OM, n=15) of patients with an oral mucocele. Cell migration ability was evaluated a scratch wound assay in SCC9 and HaCat cell lines RESULTS: E-cadherin mRNA expression in the cell lines SCC9 and HaCat was significantly reduced under hypoxia, regardless of the methylation profile, when compared to the control group. No differences in methylation profile of the E-cadherin were observed among the groups OM, PMOL and OSCC. HaCat and SCC9 presented increases in cell migration rates under hypoxia. The current study demonstrates that hypoxia reduces E-cadherin expression and increase cell migration, regardless of the methylation profile. Additionally, no differences in E-cadherin methylation patterns were observed among OM, PMOL and OSCC. Copyright © 2017 Elsevier GmbH. All rights reserved.

Ag+ alters cell growth, neurite extension, cardiomyocyte beating, and fertilized egg constriction.

PubMed

Conrad, A H; Tramp, C R; Long, C J; Wells, D C; Paulsen, A Q; Conrad, G W

1999-11-01

The Russian Space Agency uses electrochemically generated silver ions (Ag+) to purify drinking water for their space station, Mir, and their portion of the International Space Station. U.S. EPA guidelines allow 10.6 micromol x L(-1) Ag+ in human drinking water for up to 10 d. Studies correlate Ag+ exposure with tissue dysfunction in humans, rats, and mice, and with altered ion transport, skeletal muscle contraction, and embryonic cell constriction in other animal cells. Ag+ effects on cell shape change-related functions have not been assessed. Immortalized embryonic human intestinal epithelial cells, freshly explanted embryonic avian nerve cells and cardiomyocytes, and marine fertilized eggs were grown in vitro in medium containing AgNO3. Intestinal cells detach from the substratum and viable cell number decreases by 5-6 d at 5 micromol x L(-1) AgNO3, and faster at higher concentrations. Microtubules appear unaltered in adherent cells. Detached cells are nonviable. Neurite outgrowth and glial cell migration from dorsal root ganglia are inhibited by 3 d at 15 micromol x L(-1) AgNO3 or greater. Contractions stop temporarily in most cardiomyocytes by 5 min at 5 micromol x L(-1) AgNO3 or more, but some cardiomyocytes beat 3 times faster than normal at 7.5-20 micromol x L(-1) AgNO3. Picomolar Ag+ increases marine egg polar lobe constriction within an hour, even in the absence of microtubules. Ag+ alters animal cell growth and shape changes by a MT-independent mechanism. This is the first report of Ag+ effects on vertebrate neurite outgrowth, glial cell migration, or cardiomyocyte beat rate.

Photosynthetic leaf area modulates tiller bud outgrowth in sorghum: Bud outgrowth is sensitive to leaf area

DOE PAGES

Kebrom, Tesfamichael H.; Mullet, John E.

2014-12-12

Shoot branches or tillers develop from axillary buds. The dormancy versus outgrowth fates of buds depends on genetic, environmental and hormonal signals. Defoliation inhibits bud outgrowth indicating the role of leaf-derived metabolic factors such as sucrose in bud outgrowth. In this study, the sensitivity of bud outgrowth to selective defoliation was investigated. At 6 d after planting (6 DAP), the first two leaves of sorghum were fully expanded and the third was partially emerged. Therefore, the leaves were selectively defoliated at 6 DAP and the length of the bud in the first leaf axil was measured at 8 DAP. Budmore » outgrowth was inhibited by defoliation of only 2 cm from the tip of the second leaf blade. The expression of dormancy and sucrose-starvation marker genes was up-regulated and cell cycle and sucrose-inducible genes was down-regulated during the first 24 h postdefoliation of the second leaf.At 48 h, the expression of these genes was similar to controls as the defoliated plant recovers. Our results demonstrate that small changes in photosynthetic leaf area affect the propensity of tiller buds for outgrowth. Therefore, variation in leaf area and photosynthetic activity should be included when integrating sucrose into models of shoot branching.« less

ERβ1 inhibits the migration and invasion of breast cancer cells through upregulation of E-cadherin in a Id1-dependent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Yan; Ming, Jia; Xu, Yan

2015-02-06

Highlights: • Expression of ERβ1 was positively correlated with E-cadherin in breast cancer cell. • ERβ1 upregulates E-cadherin expression in breast cancer cell lines. • ERβ1 upregulates E-cadherin expression in a Id1-dependent manner. - Abstract: ERβ1 is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. It plays an important role in regulating the progression of breast cancer. However, the mechanisms of ERβ1 in tumorigenesis, metastasis and prognosis are still not fully clear. In this study, we showed that the expression of ERβ1 was positively correlated with E-cadherin expression in breast cancer cell lines. In addition, we foundmore » that ERβ1 upregulates E-cadherin expression in breast cancer cell lines. Furthermore, we also found that ERβ1 inhibits the migration and invasion of breast cancer cells and upregulated E-cadherin expression in a Id1-dependent manner. Taken together, our study provides further understanding of the molecular mechanism of ERβ1 in tumor metastasis and suggests the feasibility of developing novel therapeutic approaches to target Id1 to inhibit breast cancer metastasis.« less

N-cadherin locks left-right asymmetry by ending the leftward movement of Hensen's node cells.

PubMed

Mendes, Raquel V; Martins, Gabriel G; Cristovão, Ana M; Saúde, Leonor

2014-08-11

The stereotypic left-right (LR) asymmetric distribution of internal organs is due to an asymmetric molecular cascade in the lateral plate mesoderm (LPM) that is originated at the embryonic node. In chicken embryos, molecular asymmetries at Hensen's node are created by leftward cell movements that occur transiently. What terminates these movements, and, moreover, what is the impact of prolonging them on the LR asymmetry cascade? We show that leftward movements last longer when N-cadherin function is blocked and cease prematurely when N-cadherin is overexpressed on the right side of the node. The prolonged leftward movements lead to loss of asymmetric expression of fgf8 and nodal at the node region. This originates an abnormal expression of the asymmetric genes cer1 and snai1 in the LPM, resulting in mispositioned hearts. We conclude that N-cadherin stops the leftward cell movements and that this termination is an essential step in the establishment of LR asymmetry. Copyright © 2014 Elsevier Inc. All rights reserved.

Nonagonal cadherins: A new protein family found within the Stramenopiles.

PubMed

Fletcher, Kyle I G; van West, Pieter; Gachon, Claire M M

2016-11-15

Cadherins, a group of molecules typically associated with planar cell polarity and Wnt signalling, have been little reported outside of the animal kingdom. Here, we identify a new family of cadherins in the Stramenopiles, termed Nonagonal after their 9 transmembrane passes, which contrast to the one or seven passes found in other known cadherin families. Manual curation and experimental validation reveal two subclasses of nonagonal cadherins, depending on the number of uninterrupted extracellular cadherin (EC) modules presented. Firstly, shorter mono-exonic, unimodular, protein models, with 3 to 12 EC domains occur as duplicate paralogs in the saprotrophic Labyrinthulomycetes Aurantiochytrium limanicum and Schizochytrium aggregatum, the gastrointestinal Blastocystis hominis (Blastocystae) and as a single copy gene in the autotrophic Pelagophyte Aureococcus anophagefferens. Larger, single copy, multi-exonal, tri-modular protein models, with up to 72 EC domain in total, are found in the Oomycete genera Albugo, Phytophthora, Pythium and Eurychasma. No homolog was found in the closely related autotrophic Phaeophyceae (brown algae) or Bacillariophyceae (diatoms), nor in several genera of plant and animal pathogenic oomycetes (Aphanomyces, Saprolegnia and Hyaloperonospora). This potential absence was further investigated by synteny analysis of the genome regions flanking the cadherin gene models, which are found to be highly variable. Novel to this new cadherin family is the presence of intercalated laminin and putative carbohydrate binding in tri-modular oomycete cadherins and at the N-terminus of thraustochytrid proteins. As we were unable to detect any homologs of proteins involved in signalling pathways where other cadherin families are involved, we present a conceptual hypothesis on the function of nonagonal cadherin based around the presence of putative carbohydrate binding domains. Copyright © 2016. Published by Elsevier B.V.

Modulation of N-glycosylation by mesalamine facilitates membranous E-cadherin expression in colon epithelial cells☆

PubMed Central

Khare, Vineeta; Lang, Michaela; Dammann, Kyle; Campregher, Christoph; Lyakhovich, Alex; Gasche, Christoph

2014-01-01

Genome wide association studies have implicated intestinal barrier function genes in the pathogenesis of ulcerative colitis. One of such loci CDH1, encoding E-cadherin, a transmembrane glycoprotein with known tumor suppressor functions, is also linked to the susceptibility to colorectal cancer. Loss of membranous E-cadherin expression is common in both colitis and cancer. We have recently demonstrated that mesalamine (5-ASA); the anti-inflammatory drug used to treat ulcerative colitis, induces membranous expression of E-cadherin and increases intercellular adhesion. Using colorectal cancer epithelial cells with aberrant E-cadherin expression, we investigated the mechanism underlying such an effect of 5-ASA. Post-translational modification of E-cadherin glycosylation was analyzed by biotin/streptavidin detection of sialylated glycoproteins. GnT-III (N-acetylglucosaminyltransferase III) expression was assessed by qRT-PCR, Western blot and immunofluorescence. GnT-III activity was analyzed by reactivity with E-4/L-4-PHA. Expression, localization and interaction of E-cadherin and β-catenin were analyzed by Western blot, immunocytochemistry and RNA interference. 5-ASA activity modulated E-cadherin glycosylation and increased both mRNA and protein levels of GnT-III and its activity as detected by increased E4-lectin reactivity. Intestinal APCMin polyps in mice showed low expression of GnT-III and 5-ASA was effective in increasing its expression. The data demonstrated that remodeling of glycans by GnT-III mediated bisect glycosylation, contributes to the membranous retention of E-cadherin by 5-ASA; facilitating intercellular adhesion. Induction of membranous expression of E-cadherin by 5-ASA is a novel mechanism for mucosal healing in colitis that might impede tumor progression by modulation of GnT-III expression. PMID:24184502

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

Neurite outgrowth of murine cerebellar granule cells can be enhanced by aniracetam with or without alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA).

PubMed

Fushiki, S; Matsumoto, K; Nagata, A

1995-10-27

To assess the neurotrophic effects of a nootropic drug, aniracetam, we studied neurite extension of mouse cerebellar granule cells in culture with low or with high K+ under different combinations of drugs and then immunohistochemically stained the cells with an antibody against L1, a neural cell adhesion molecule on cerebellar granule cells. Quantitative analyses using parameters of the total neurite length, maximal neurite length and number of branches disclosed that aniracetam, in the presence of high K+ and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), significantly enhanced neurite extension of cultured granule neurons. Aniracetam alone also stimulated neurite extension of cerebellar granule cells at a longer period of culture with low K+ showing a bell-shaped dose response curve with maximal effects at 10 microM. Aniracetam may influence remodeling of the neural network after injury.

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.

Arf6 guanine-nucleotide exchange factor, cytohesin-2, interacts with actinin-1 to regulate neurite extension.

PubMed

Torii, Tomohiro; Miyamoto, Yuki; Nakamura, Kazuaki; Maeda, Masahiro; Yamauchi, Junji; Tanoue, Akito

2012-09-01

Proper regulation of morphological changes in neuronal cells is essential for their differentiation. Complex signaling mechanisms mediate a variety of morphological changes such as formation of neurites. It is well established that a number of small GTPases control neurite behavior before the connection with the target tissue. However, their regulatory mechanisms remain to be fully understood. Here, we show that the Arf6 guanine-nucleotide exchange factor (GEF), cytohesin-2 (CYTH2), interacts with the cytoskeletal protein actinin-1 (ACTN1) and regulates neurite extension in N1E-115 cells used as the model. Knockdown of ACTN1, as well as that of CYTH2, in cells inhibits cellular Arf6 activity and neurite extension. The C-terminal polybasic region of CYTH2 participates in interacting directly with the EFh2 domain of ACTN1. Expression of CYTH2 mutant deficient of the EFh2 domain in cells also inhibits Arf6 activation and neurite extension. Furthermore, FRET analysis detects that the respective interactive region peptides, tagged with cell-permeable short peptides, greatly decrease Arf6 activation at growth cones in a time-dependent manner. Collectively, the signaling through CYTH2 and ACTN1 properly regulates neurite extension in N1E-115 cells, demonstrating the unexpected interaction of CYTH2 and ACTN1 in the regulation of cellular Arf6 activity involved in neurite extension. Copyright © 2012 Elsevier Inc. All rights reserved.

Dipentylammonium Binds to the Sigma-1 Receptor and Protects Against Glutamate Toxicity, Attenuates Dopamine Toxicity and Potentiates Neurite Outgrowth in Various Cultured Cell Lines.

PubMed

Brimson, James M; Safrany, Stephen T; Qassam, Heider; Tencomnao, Tewin

2018-03-27

Alzheimer's disease is a neurodegenerative disease that affects 44 million people worldwide, costing the world $605 billion to care for those affected not taking into account the physical and psychological costs for those who care for Alzheimer's patients. Dipentylammonium is a simple amine, which is structurally similar to a number of other identified sigma-1 receptor ligands with high affinities such as (2R-trans)-2butyl-5-heptylpyrrolidine, stearylamine and dodecylamine. This study investigates whether dipentylammonium is able to provide neuroprotective effects similar to those of sigma-1 receptor agonists such as PRE-084. Here we identify dipentylammonium as a sigma-1 receptor ligand with nanomolar affinity. We have found that micromolar concentrations of dipentylammonium protect from glutamate toxicity and prevent NFκB activation in HT-22 cells. Micromolar concentrations of dipentylammonium also protect stably expressing amyloid precursor protein Swedish mutant (APP/Swe) Neuro2A cells from toxicity induced by 150 μM dopamine, suggesting that dipentylammonium may be useful for the treatment of Parkinsonian symptoms in Alzheimer's patients which are often associated with a more rapid deterioration of cognitive and physical ability. Finally, we found that low micromolar concentrations of dipentylammonium could out preform known sigma-1 receptor agonist PRE-084 in potentiating neurite outgrowth in Neuro2A cells, further suggesting that dipentylammonium has a potential use in the treatment of neurodegenerative diseases and could be acting through the sigma-1 receptor.

A dual role for the RhoGEF Ephexin5 in regulation of dendritic spine outgrowth

PubMed Central

Hamilton, AM; Lambert, JT; Parajuli, LK; Vivas, O; Park, DK; Stein, IS; Jahncke, JN; Greenberg, ME; Margolis, SS; Zito, K

2017-01-01

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines. PMID:28185854

A dual role for the RhoGEF Ephexin5 in regulation of dendritic spine outgrowth.

PubMed

Hamilton, A M; Lambert, J T; Parajuli, L K; Vivas, O; Park, D K; Stein, I S; Jahncke, J N; Greenberg, M E; Margolis, S S; Zito, K

2017-04-01

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines. Copyright © 2017 Elsevier Inc. All rights reserved.

DE-Cadherin Is Required for Intercellular Motility during Drosophila Oogenesis

PubMed Central

Niewiadomska, Paulina; Godt, Dorothea; Tepass, Ulrich

1999-01-01

Cadherins are involved in a variety of morphogenetic movements during animal development. However, it has been difficult to pinpoint the precise function of cadherins in morphogenetic processes due to the multifunctional nature of cadherin requirement. The data presented here indicate that homophilic adhesion promoted by Drosophila E-cadherin (DE-cadherin) mediates two cell migration events during Drosophila oogenesis. In Drosophila follicles, two groups of follicle cells, the border cells and the centripetal cells migrate on the surface of germline cells. We show that the border cells migrate as an epithelial patch in which two centrally located cells retain epithelial polarity and peripheral cells are partially depolarized. Both follicle cells and germline cells express DE-cadherin, and border cells and centripetal cells strongly upregulate the expression of DE-cadherin shortly before and during their migration. Removing DE-cadherin from either the follicle cells or the germline cells blocks migration of border cells and centripetal cells on the surface of germline cells. The function of DE-cadherin in border cells appears to be specific for migration as the formation of the border cell cluster and the adhesion between border cells are not disrupted in the absence of DE-cadherin. The speed of migration depends on the level of DE-cadherin expression, as border cells migrate more slowly when DE-cadherin activity is reduced. Finally, we show that the upregulation of DE-cadherin expression in border cells depends on the activity of the Drosophila C/EBP transcription factor that is essential for border cell migration. PMID:9971747

Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells.

PubMed

Xu, Rui; Zhang, Yujie; Gu, Luo; Zheng, Jianchao; Cui, Jie; Dong, Jing; Du, Jun

2015-01-01

E-cadherin internalization facilitates dissolution of adherens junctions and promotes tumor cell epithelial-mesenchymal transition (EMT) and migration. Our previous results have shown that Arf6 exerts pro-migratory action in breast cancer cells after EGF stimulation. Despite the fact that EGF signaling stimulates EMT of breast cancer cells, the effect of Arf6 on internalization of E-cadherin of breast cancer cells under EGF treatment remains to be determined. Here, we showed that EGF dose-dependently stimulated E-cadherin internalization by MCF-7 cells with the maximal effect at 50 ng/ml. Meanwhile, EGF treatment markedly increased Arf6 activation. Arf6 was involved in complexes of E-cadherin, and more E-cadherin was pulled down with Arf6 when the activity of the latter was increased. Immunoblotting and immunofluorescence assays showed that transfection breast cancer cells with Arf6-T27N or Arf6 siRNA suppressed EGF-induced E-cadherin internalization. Taken together, our study demonstrated that Arf6 activation plays a potential role in EGF-induced E-cadherin internalization, providing new mechanism underlying the effect of Arf6 on promoting breast cancer cell metastasis.

Cytoplasmic p21Cip1/WAF1 regulates neurite remodeling by inhibiting Rho-kinase activity

PubMed Central

Tanaka, Hiroyuki; Yamashita, Toshihide; Asada, Minoru; Mizutani, Shuki; Yoshikawa, Hideki; Tohyama, Masaya

2002-01-01

p21Cip1/WAF1 has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21Cip1/WAF1 is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21Cip1/WAF1 lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21Cip1/WAF1 forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21Cip1/WAF1 is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21Cip1/WAF1 may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions. PMID:12119358

E-cadherin-mediated force transduction signals regulate global cell mechanics

PubMed Central

Muhamed, Ismaeel; Wu, Jun; Sehgal, Poonam; Kong, Xinyu; Tajik, Arash; Wang, Ning

2016-01-01

ABSTRACT This report elucidates an E-cadherin-based force-transduction pathway that triggers changes in cell mechanics through a mechanism requiring epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), and the downstream formation of new integrin adhesions. This mechanism operates in addition to local cytoskeletal remodeling triggered by conformational changes in the E-cadherin-associated protein α-catenin, at sites of mechanical perturbation. Studies using magnetic twisting cytometry (MTC), together with traction force microscopy (TFM) and confocal imaging identified force-activated E-cadherin-specific signals that integrate cadherin force transduction, integrin activation and cell contractility. EGFR is required for the downstream activation of PI3K and myosin-II-dependent cell stiffening. Our findings also demonstrated that α-catenin-dependent cytoskeletal remodeling at perturbed E-cadherin adhesions does not require cell stiffening. These results broaden the repertoire of E-cadherin-based force transduction mechanisms, and define the force-sensitive signaling network underlying the mechano-chemical integration of spatially segregated adhesion receptors. PMID:26966187

Leptospira interrogans Binds to Cadherins

PubMed Central

Evangelista, Karen; Franco, Ricardo; Schwab, Andrew; Coburn, Jenifer

2014-01-01

Leptospirosis, caused by pathogenic species of Leptospira, is the most widespread zoonosis and has emerged as a major public health problem worldwide. The adhesion of pathogenic Leptospira to host cells, and to extracellular matrix (ECM) components, is likely to be necessary for the ability of leptospires to penetrate, disseminate and persist in mammalian host tissues. Previous work demonstrated that pathogenic L. interrogans binds to host cells more efficiently than to ECM. Using two independent screening methods, mass spectrometry and protein arrays, members of the cadherin family were identified as potential L. interrogans receptors on mammalian host surfaces. We focused our investigation on vascular endothelial (VE)-cadherin, which is widely expressed on endothelia and is primarily responsible for endothelial cell-cell adhesion. Monolayers of EA.hy926 and HMEC-1 endothelial cells produce VE-cadherin, bind L. interrogans in vitro, and are disrupted upon incubation with the bacteria, which may reflect the endothelial damage seen in vivo. Dose-dependent and saturable binding of L. interrogans to the purified VE-cadherin receptor was demonstrated and pretreatment of purified receptor or endothelial cells with function-blocking antibody against VE-cadherin significantly inhibited bacterial attachment. The contribution of VE-cadherin to leptospiral adherence to host endothelial cell surfaces is biologically significant because VE-cadherin plays an important role in maintaining the barrier properties of the vasculature. Attachment of L. interrogans to the vasculature via VE-cadherin may result in vascular damage, facilitating the escape of the pathogen from the bloodstream into different tissues during disseminated infection, and may contribute to the hemorrhagic manifestations of leptospirosis. This work is first to describe a mammalian cell surface protein as a receptor for L. interrogans. PMID:24498454

Immunohistochemical localization of cell adhesion molecule epithelial cadherin in human arachnoid villi and meningiomas.

PubMed

Tohma, Y; Yamashima, T; Yamashita, J

1992-04-01

Cadherins are a family of intercellular glycoproteins responsible for calcium-dependent cell adhesion and are currently divided into four types: epithelial (E), neuronal (N), placental (P), and vascular (V). Since cadherins are known to be indispensable for not only morphogenesis in the embryo but also maintenance of tumor cell nest, we examined the expression of E-cadherin in 31 meningiomas (11 syncytial, 12 transitional, 8 fibroblastic) and 3 arachnoid villi by immunoblot and immunohistochemical analyses. In the immunoblot analysis, E-cadherin was detected at the main band of Mr 124,000 in all of the arachnoid villi, as well as syncytial and transitional types of meningiomas, but not in the fibroblastic type. The immunohistochemical examination showed that E-cadherin was expressed at the cell borders of syncytial and transitional types, but the expression was absent in the fibroblastic type. Immunoelectron microscopy showed that E-cadherin was localized at the intermediate junctions in arachnoid villi, while it was detected diffusely at the cell surface in meningiomas. It is suggested from these data that the expression of E-cadherin might be closely related to the differentiation and organogenesis of meningioma cells.

Melatonin induces neuritogenesis at early stages in N1E-115 cells through actin rearrangements via activation of protein kinase C and Rho-associated kinase.

PubMed

Bellon, Alfredo; Ortíz-López, Leonardo; Ramírez-Rodríguez, Gerardo; Antón-Tay, Fernando; Benítez-King, Gloria

2007-04-01

Melatonin increases neurite formation in N1E-115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) activation. Microfilament rearrangement is also a necessary process in growth cone formation during neurite outgrowth. In this work, we studied the effect of melatonin on microfilament rearrangements present at early stages of neurite formation and the possible participation of PKC and the Rho-associated kinase (ROCK), which is a downstream kinase in the PKC signaling pathway. The results showed that 1 nm melatonin increased both the number of cells with filopodia and with long neurites. Similar results were obtained with the PKC activator phorbol 12-myristate 13-acetate (PMA). Both melatonin and PMA increased the quantity of filamentous actin. In contrast, the PKC inhibitor bisindolylmaleimide abolished microfilament organization elicited by either melatonin or PMA, while the Rho inhibitor C3, or the ROCK inhibitor Y27632, abolished the bipolar neurite morphology of N1E-115 cells. Instead, these inhibitors prompted neurite ramification. ROCK activity measured in whole cell extracts and in N1E-115 cells was increased in the presence of melatonin and PMA. The results indicate that melatonin increases the number of cells with immature neurites and suggest that these neurites can be susceptible to differentiation by incoming extracellular signals. Data also indicate that PKC and ROCK are involved at initial stages of neurite formation in the mechanism by which melatonin recruits cells for later differentiation.

Growth of neurites toward neurite- neurite contact sites increases synaptic clustering and secretion and is regulated by synaptic activity.

PubMed

Cove, Joshua; Blinder, Pablo; Abi-Jaoude, Elia; Lafrenière-Roula, Myriam; Devroye, Luc; Baranes, Danny

2006-01-01

The integrative properties of dendrites are determined by several factors, including their morphology and the spatio-temporal patterning of their synaptic inputs. One of the great challenges is to discover the interdependency of these two factors and the mechanisms which sculpt dendrites' fine morphological details. We found a novel form of neurite growth behavior in neuronal cultures of the hippocampus and cortex, when axons and dendrites grew directly toward neurite-neurite contact sites and crossed them, forming multi-neurite intersections (MNIs). MNIs were found at a frequency higher than obtained by computer simulations of randomly distributed dendrites, involved many of the dendrites and were stable for days. They were formed specifically by neurites originating from different neurons and were extremely rare among neurites of individual neurons or among astrocytic processes. Axonal terminals were clustered at MNIs and exhibited higher synaptophysin content and release capability than in those located elsewhere. MNI formation, as well as enhancement of axonal terminal clustering and secretion at MNIs, was disrupted by inhibitors of synaptic activity. Thus, convergence of axons and dendrites to form MNIs is a non-random activity-regulated wiring behavior which shapes dendritic trees and affects the location, clustering level and strength of their presynaptic inputs.

The Integrated Role of Wnt/β-Catenin, N-Glycosylation, and E-Cadherin-Mediated Adhesion in Network Dynamics

PubMed Central

Vargas, Diego A.; Sun, Meng; Sadykov, Khikmet; Kukuruzinska, Maria A.; Zaman, Muhammad H.

2016-01-01

The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene, DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability

A low-cost microwell device for high-resolution imaging of neurite outgrowth in 3D

NASA Astrophysics Data System (ADS)

Ren, Yuan; Mlodzianoski, Michael J.; Cheun Lee, Aih; Huang, Fang; Suter, Daniel M.

2018-06-01

Objective. Current neuronal cell culture is mostly performed on two-dimensional (2D) surfaces, which lack many of the important features of the native environment of neurons, including topographical cues, deformable extracellular matrix, and spatial isotropy or anisotropy in three dimensions. Although three-dimensional (3D) cell culture systems provide a more physiologically relevant environment than 2D systems, their popularity is greatly hampered by the lack of easy-to-make-and-use devices. We aim to develop a widely applicable 3D culture procedure to facilitate the transition of neuronal cultures from 2D to 3D. Approach. We made a simple microwell device for 3D neuronal cell culture that is inexpensive, easy to assemble, and fully compatible with commonly used imaging techniques, including super-resolution microscopy. Main results. We developed a novel gel mixture to support 3D neurite regeneration of Aplysia bag cell neurons, a system that has been extensively used for quantitative analysis of growth cone dynamics in 2D. We found that the morphology and growth pattern of bag cell growth cones in 3D culture closely resemble the ones of growth cones observed in vivo. We demonstrated the capability of our device for high-resolution imaging of cytoskeletal and signaling proteins as well as organelles. Significance. Neuronal cell culture has been a valuable tool for neuroscientists to study the behavior of neurons in a controlled environment. Compared to 2D, neurons cultured in 3D retain the majority of their native characteristics, while offering higher accessibility, control, and repeatability. We expect that our microwell device will facilitate a wider adoption of 3D neuronal cultures to study the mechanisms of neurite regeneration.

N-cadherin{sup +} HSCs in fetal liver exhibit higher long-term bone marrow reconstitution activity than N-cadherin{sup -} HSCs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toyama, Hirofumi; Arai, Fumio; Hosokawa, Kentaro

Highlights: Black-Right-Pointing-Pointer High N-cad expression was detected in E12.5 mouse FL LT-HSCs (EPCR{sup +} LSK cells). Black-Right-Pointing-Pointer Immunohistochemically, N-cad{sup +} HSCs co-localized with sinusoidal ECs (Lyve-1{sup +} cells) in E12.5 FL, but these gradually detached in E15.5 and E18.5 FL. Black-Right-Pointing-Pointer N-cad{sup +} LSK cells in E12.5 FL exhibited higher LTR activity versus N-cad{sup -} LSK cells, which decreased in E15.5 and E18.5. Black-Right-Pointing-Pointer N-cad expression may confer high LTR activity to HSCs by facilitating interactions with the perisinusoidal niche in FL. -- Abstract: Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment known as the stem cell niche.more » The regulation of HSCs in fetal liver (FL) and their niche, however, remains to be elucidated. In this study, we investigated the role of N-cadherin (N-cad) in the maintenance of HSCs during FL hematopoiesis. By using anti-N-cad antibodies (Abs) produced by our laboratory, we detected high N-cad expression in embryonic day 12.5 (E12.5) mouse FL HSCs, but not in E15.5 and E18.5 FL. Immunofluorescence staining revealed that N-cad{sup +}c-Kit{sup +} and N-cad{sup +} endothelial protein C receptor (EPCR){sup +} HSCs co-localized with Lyve-1{sup +} sinusoidal endothelial cells (ECs) in E12.5 FL and that some of these cells also expressed N-cad. However, N-cad{sup +} HSCs were also observed to detach from the perisinusoidal niche at E15.5 and E18.5, concomitant with a down-regulation of N-cad and an up-regulation of E-cadherin (E-cad) in hepatic cells. Moreover, EPCR{sup +} long-term (LT)-HSCs were enriched in the N-cad{sup +}Lin{sup -}Sca-1{sup +}c-Kit{sup +} (LSK) fraction in E12.5 FL, but not in E15.5 or E18.5 FL. In a long-term reconstitution (LTR) activity assay, higher engraftment associated with N-cad{sup +} LSK cells versus N-cad{sup -} LSK cells in E12.5 FL when transplanted into lethally irradiated recipient mice. However

CHOLINE PARTIALLY PREVENTS THE IMPACT OF ETHANOL ON THE LIPID RAFT DEPENDENT FUNCTIONS OF L1 CELL ADHESION MOLECULE

PubMed Central

Tang, Ningfeng; Bamford, Penny; Jones, Jace; He, Min; Kane, Maureen A.; Mooney, Sandra M.; Bearer, Cynthia F.

2014-01-01

Background Fetal Alcohol Spectrum Disorder, the leading known cause of mental retardation, is caused by alcohol exposure during pregnancy. One mechanism of ethanol teratogenicity is the disruption of the function of L1 cell adhesion molecule (L1). These functions include enhancement of neurite outgrowth, trafficking through lipid rafts, and signal transduction. Recent data have shown that choline supplementation of rat pups reduces the effects of ethanol on neurobehavior. We sought to determine if choline could prevent the effect of ethanol on L1 function using a simple experimental system. Methods Cerebellar granule neurons (CGN) from postnatal day 6 rat pups were cultured with and without supplemental choline, and the effects on L1 signaling, lipid raft distribution and neurite outgrowth were measured in the presence or absence of ethanol. Results Choline significantly reduced the effect of ethanol on L1 signaling, the distribution of L1 in lipid rafts and L1 mediated neurite outgrowth. However, choline supplemented ethanol exposed cultures remained significantly different than controls. Conclusions Choline pretreatment of CGN significantly reduces the disruption of L1 function by ethanol, but does not completely return L1 function to baseline. This experimental system will enable discovery of the mechanism of the neuroprotective effect of choline. PMID:25421509

Paradoxical expression of E-cadherin in prostatic bone metastases.

PubMed

Bryden, A A; Freemont, A J; Clarke, N W; George, N J

1999-12-01

To determine whether the calcium-dependent cell adhesion molecule E-cadherin is expressed in metastatic deposits of prostate cancer in bone. Ten bone biopsies containing metastatic deposits of untreated prostatic cancer were obtained and immunohistochemically stained for E-cadherin with the monoclonal antibody HECD-1, using the streptavidin-biotin complex technique. Benign prostatic tissue was used as the control. Of the 10 specimens, nine showed positive expression of E-cadherin, graded as strong in four. E-cadherin expression was strongest in well-differentiated metastases and decreased with increasing tumour grade. In some specimens there were mixed patterns of expression. E-cadherin is strongly expressed in prostatic bone metastases and the degree of expression appears to reflect local tumour grade. This suggests that loss of E-cadherin expression may not be critically linked to metastatic potential.

Distinct 3-O-Sulfated Heparan Sulfate Modification Patterns Are Required for kal-1−Dependent Neurite Branching in a Context-Dependent Manner in Caenorhabditis elegans

PubMed Central

Tecle, Eillen; Diaz-Balzac, Carlos A.; Bülow, Hannes E.

2013-01-01

Heparan sulfate (HS) is an unbranched glycosaminoglycan exhibiting substantial molecular diversity due to multiple, nonuniformly introduced modifications, including sulfations, epimerization, and acetylation. HS modifications serve specific and instructive roles in neuronal development, leading to the hypothesis of a HS code that regulates nervous system patterning. Although the in vivo roles of many of the HS modifications have been investigated, very little is known about the function of HS 3-O-sulfation in vivo. By examining patterning of the Caenorhabditis elegans nervous system in loss of function mutants of the two 3-O-sulfotransferases, hst-3.1 and hst-3.2, we found HS 3-O-sulfation to be largely dispensable for overall neural development. However, generation of stereotypical neurite branches in hermaphroditic-specific neurons required hst-3.1, hst-3.2, as well as an extracellular cell adhesion molecule encoded by kal-1, the homolog of Kallmann Syndrome associated gene 1/anosmin-1. In contrast, kal-1−dependent neurite branching in AIY neurons required catalytic activity of hst-3.2 but not hst-3.1. The context-dependent requirement for hst-3.2 and hst-3.1 indicates that both enzymes generate distinct types of HS modification patterns in different cell types, which regulate kal-1 to promote neurite branching. We conclude that HS 3-O-sulfation does not play a general role in establishing the HS code in C. elegans but rather plays a specialized role in a context-dependent manner to establish defined aspects of neuronal circuits. PMID:23451335

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

Ependymin as a substrate for outgrowth of axons from cultured explants of goldfish retina.

PubMed

Schmidt, J T; Schmidt, R; Lin, W C; Jian, X Y; Stuermer, C A

1991-01-01

Ependymin, a prominent protein of the brain's extracellular fluid (ECF) was previously implicated in the consolidation of memory and in the activity-driven sharpening of the retinotectal projection. Because both these phenomena probably involve the growth and elaboration of appropriate synapses, we have tested whether ependymin can serve as a substrate for the growth of axons from goldfish retinal ganglion cells in a culture assay. Ependymin (Ep), laminin (LAM), polylysine (PL), and Concanavalin A (Con A) were plated on glass coverslips either uniformly or in striped patterns. Ep alone, either soluble or partly polymerized (by dropping calcium concentration and pH), was a good substrate for axonal outgrowth, as good or better than PL and Con A, but not as good as LAM. Neurites grew faster on LAM (71 microns/h) than on Ep (32 microns/h) or on PL (22 microns/h). Fasciculation was low on LAM, intermediate on Ep, and highest on PL. In exclusive side-by-side stripe assays, axons preferred LAM over Ep, but gave weak or no preference for Ep over Con A or PL. With stripes of LAM + Ep alongside pure LAM, the axons preferred the mixture of LAM + Ep. When antibodies to Ep were plated in stripes over continuous Ep substrate, the axons avoided the antibody-blocked stripes and grew on the Ep stripes. Antibodies to Ep did not, however, block growth on laminin substrates, nor did antibodies to LAM block growth on Ep. Dot blots and western blots showed very little cross recognition between the antibodies. Ependymin is a good substrate for neurite outgrowth, which is normally present in ECF, and adhesion to Ep is independent of LAM and possibly additive to it.

Constitutive Overexpression of the Basic Helix-Loop-Helix Nex1/MATH-2 Transcription Factor Promotes Neuronal Differentiation of PC12 Cells and Neurite Regeneration

PubMed Central

Uittenbogaard, Martine; Chiaramello, Anne

2009-01-01

Elucidation of the intricate transcriptional pathways leading to neural differentiation and the establishment of neuronal identity is critical to the understanding and design of therapeutic approaches. Among the important players, the basic helix-loop-helix (bHLH) transcription factors have been found to be pivotal regulators of neurogenesis. In this study, we investigate the role of the bHLH differentiation factor Nex1/MATH-2 in conjunction with the nerve growth factor (NGF) signaling pathway using the rat phenochromocytoma PC12 cell line. We report that the expression of Nex1 protein is induced after 5 hr of NGF treatment and reaches maximal levels at 24 hr, when very few PC12 cells have begun extending neurites and ceased cell division. Furthermore, our study demonstrates that Nex1 has the ability to trigger neuronal differentiation of PC12 cells in the absence of neurotrophic factor. We show that Nex1 plays an important role in neurite outgrowth and has the capacity to regenerate neurite outgrowth in the absence of NGF. These results are corroborated by the fact that Nex1 targets a repertoire of distinct types of genes associated with neuronal differentiation, such as GAP-43, βIII-tubulin, and NeuroD. In addition, our findings show that Nex1 up-regulates the expression of the mitotic inhibitor p21WAF1, thus linking neuronal differentiation to cell cycle withdrawal. Finally, our studies show that overexpression of a Nex1 mutant has the ability to block the execution of NGF-induced differentiation program, suggesting that Nex1 may be an important effector of the NGF signaling pathway. PMID:11782967

Consequences of Neurite Transection In Vitro

PubMed Central

Cengiz, Nurettin; Erdoğan, Ender; Him, Aydın; Oğuz, Elif Kaval

2012-01-01

Abstract In order to quantify degenerative and regenerative changes and analyze the contribution of multiple factors to the outcome after neurite transection, we cultured adult mouse dorsal root ganglion neurons, and with a precise laser beam, we transected the nerve fibers they extended. Cell preparations were continuously visualized for 24 h with time-lapse microscopy. More distal cuts caused a more elongated field of degeneration, while thicker neurites degenerated faster than thinner ones. Transected neurites degenerated more if the uncut neurites of the same neuron simultaneously degenerated. If any of these uncut processes regenerated, the transected neurites underwent less degeneration. Regeneration of neurites was limited to distal cuts. Unipolar neurons had shorter regeneration than multipolar ones. Branching slowed the regenerative process, while simultaneous degeneration of uncut neurites increased it. Proximal lesions, small neuronal size, and extensive and rapid neurite degeneration were predictive of death of an injured neuron, which typically displayed necrotic rather than apoptotic form. In conclusion, this in vitro model proved useful in unmasking many new aspects and correlates of mechanically-induced neurite injury. PMID:20121423

Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion

PubMed Central

Chitaev, Nikolai A.; Troyanovsky, Sergey M.

1997-01-01

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg1-Thr170) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca2+-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca2+-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion. PMID:9214392

A herpes simplex viral vector expressing green fluorescent protein can be used to visualize morphological changes in high-density neuronal culture

PubMed Central

Falk, Torsten; Strazdas, Lori A.; Borders, Rebecca S.; Kilani, Ramsey K.; Yool, Andrea J.

2010-01-01

High-density cultures of mammalian neurons offer a model system for studies of brain development, but the morphological features of individual neurons is difficult to ascertain. We show that a herpes virus vector expressing a bioluminescent protein allows detailed morphometric analyses of living neurons in complex culture environments. Expression of enhanced green fluorescent protein (eGFP) was constitutively driven in neurons using the herpes simplex virus amplicon system. This system allowed us to make novel observations regarding development in high-density cultures from rat hippocampus and cerebellum. After the phase of initial neurite outgrowth, maturing neurons continue to show rapid remodeling of the neurite branches (0.79 ± 0.11 μm/h per neurite; mean ± SEM, n=8), and displacement of the soma within the neurite arbor (1.35 ± 0.74 μm/h). These results demonstrate that a substantial capacity for morphological plasticity persists in maturing mammalian CNS neurons after cessation of net neurite outgrowth in early development. PMID:20811504

Choline partially prevents the impact of ethanol on the lipid raft dependent functions of l1 cell adhesion molecule.

PubMed

Tang, Ningfeng; Bamford, Penny; Jones, Jace; He, Min; Kane, Maureen A; Mooney, Sandra M; Bearer, Cynthia F

2014-11-01

Fetal alcohol spectrum disorder, the leading known cause of mental retardation, is caused by alcohol exposure during pregnancy. One mechanism of ethanol (EtOH) teratogenicity is the disruption of the functions of L1 cell adhesion molecule (L1). These functions include enhancement of neurite outgrowth, trafficking through lipid rafts, and signal transduction. Recent data have shown that choline supplementation of rat pups reduces the effects of EtOH on neurobehavior. We sought to determine whether choline could prevent the effect of EtOH on L1 function using a simple experimental system. Cerebellar granule neurons (CGN) from postnatal day 6 rat pups were cultured with and without supplemental choline, and the effects on L1 signaling, lipid raft distribution, and neurite outgrowth were measured in the presence or absence of EtOH. Choline significantly reduced the effect of EtOH on L1 signaling, the distribution of L1 in lipid rafts and L1-mediated neurite outgrowth. However, choline supplemented EtOH-exposed cultures remained significantly different than controls. Choline pretreatment of CGN significantly reduces the disruption of L1 function by EtOH, but does not completely return L1 function to baseline. This experimental system will enable discovery of the mechanism of the neuroprotective effect of choline. Copyright © 2014 by the Research Society on Alcoholism.

Cytoplasmic and nuclear localization of cadherin in honey bee (Apis mellifera L.) gonads.

PubMed

Florecki, Mônica M; Hartfelder, Klaus

2011-01-01

Cadherins are crucial molecules mediating cell-cell interactions between somatic and germline cells in insect and mammalian male and female gonads. We analysed the presence and localization of cadherins in ovaries of honeybee queens and in testes of drones. Transcripts representing two classical cadherins, E-cadherin (shotgun) and N-cadherin, as well as three protocadherins (Starry night, Fat and Fat-like) were detected in gonads of both sexes. Pan-cadherin antibodies, which most probably detect a honeybee N-cadherin, were used in immunolocalization analyses. In the germarium of ovarioles, cadherin-IR (cadherin immunoreactivity) was evidenced as homogeneously distributed in the cytoplasm and as nuclear foci, in both germline and somatic cells. It was also detected in polyfusomes and ring canals. In testiolar tubules, cadherin-IR showed a cytoplasmic and nuclear distributon alike in ovaries. The unexpected nuclear localization and cytoplasmic distribution in ovaries and testes were corroborated by immunogold electron microscopy, which revealed cadherin aggregates associated with electron-dense nuclear structures. With respect to cadherin localization, the honeybee differs from Drosophila, the model for gametogenesis in insects, raising the question as to how differences among solitary and social species may be built into and generated from the general architecture of polytrophic meroistic ovaries. It also indicates the possibility of divergent roles for cadherin in the functional architecture of insect gonads, in general, especially in taxa with high reproductive output.

3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture

PubMed Central

Lipton, Jack W.; Tolod, Emeline G.; Thompson, Valerie B.; Pei, Lin; Paumier, Katrina L.; Terpstra, Brian T.; Lynch, Kaari A.; Collier, Timothy J.; Sortwell, Caryl E.

2008-01-01

Summary The current study examined whether modest concentrations of MDMA could increase the survival and/or neurite outgrowth of fetal midbrain dopamine (DA) neurons in vitro since increased DA neurite outgrowth has been previously observed in vivo from prenatal exposure. MDMA concentrations in fetal brain were quantified to determine relevant in vivo concentrations to employ in vitro. A dose-response study in vitro demonstrated that MDMA, at concentrations observed in vivo, resulted in increased, DA-specific, neuron survival. Higher doses resulted in nonspecific neurotoxicity. MDMA application immediately after culture establishment resulted in greater survival than delayed application, however both were superior to control. MDMA significantly increased the expression of the slc6a3 gene (dopamine transporter; DAT) in culture. Co-application of the DAT reuptake inhibitor methylphenidate (MPH) with MDMA attenuated this effect. Progressive reductions in MPH concentrations restored the MDMA-induced survival effect. This suggests that MDMA’s action at DAT mediates the survival effect. Neurite density per neuron was unaffected by MDMA in vitro suggesting that MDMA promotes DA neuron survival but not neurite outgrowth in culture. Finally, animals prenatally exposed to MDMA and examined on postnatal day 35 showed an increase in tyrosine hydroxylase-positive (TH+) neurons in the substantia nigra but not in the ventral tegmental area. These data suggest that during development, MDMA can increase the survival of DA neurons through its action at its transporter. Understanding how MDMA increases DA neuron survival may provide insight into normal DA neuron loss during development. PMID:18655796

Downregulation of P-cadherin expression in hepatocellular carcinoma induces tumorigenicity

PubMed Central

Bauer, Richard; Valletta, Daniela; Bauer, Karin; Thasler, Wolfgang E; Hartmann, Arndt; Müller, Martina; Reichert, Torsten E; Hellerbrand, Claus

2014-01-01

P-cadherin is a major contributor to cell-cell adhesion in epithelial tissues, playing pivotal roles in important morphogenetic and differentiation processes and in maintaining tissue integrity and homeostasis. Alterations of P-cadherin expression have been observed during the progression of several carcinomas where it appears to act as tumor suppressive or oncogenic in a context-dependent manner. Here, we found a significant downregulation of P-cadherin in hepatocellular carcinoma (HCC) cell lines and tissues compared to primary human hepatocytes and non-malignant liver tissues. Combined immunohistochemical analysis of a tissue microarray containing matched pairs of HCC tissue and corresponding non-tumorous liver tissue of 69 patients confirmed reduced P-cadherin expression in more than half of the cases. In 35 human HCC tissues, the P-cadherin immunosignal was completely lost which correlated with tumor staging and proliferation. Also in vitro, P-cadherin suppression in HCC cells via siRNA induced proliferation compared to cells transfected with control-siRNA. In summary, downregulation of P-cadherin expression appears to induce tumorigenicity in HCC. Therefore, P-cadherin expression may serve as a prognostic marker and therapeutic target of this highly aggressive tumor. PMID:25337260

Downregulation of P-cadherin expression in hepatocellular carcinoma induces tumorigenicity.

PubMed

Bauer, Richard; Valletta, Daniela; Bauer, Karin; Thasler, Wolfgang E; Hartmann, Arndt; Müller, Martina; Reichert, Torsten E; Hellerbrand, Claus

2014-01-01

P-cadherin is a major contributor to cell-cell adhesion in epithelial tissues, playing pivotal roles in important morphogenetic and differentiation processes and in maintaining tissue integrity and homeostasis. Alterations of P-cadherin expression have been observed during the progression of several carcinomas where it appears to act as tumor suppressive or oncogenic in a context-dependent manner. Here, we found a significant downregulation of P-cadherin in hepatocellular carcinoma (HCC) cell lines and tissues compared to primary human hepatocytes and non-malignant liver tissues. Combined immunohistochemical analysis of a tissue microarray containing matched pairs of HCC tissue and corresponding non-tumorous liver tissue of 69 patients confirmed reduced P-cadherin expression in more than half of the cases. In 35 human HCC tissues, the P-cadherin immunosignal was completely lost which correlated with tumor staging and proliferation. Also in vitro, P-cadherin suppression in HCC cells via siRNA induced proliferation compared to cells transfected with control-siRNA. In summary, downregulation of P-cadherin expression appears to induce tumorigenicity in HCC. Therefore, P-cadherin expression may serve as a prognostic marker and therapeutic target of this highly aggressive tumor.

N-cadherin adhesive interactions modulate matrix mechanosensing and fate commitment of mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Cosgrove, Brian D.; Mui, Keeley L.; Driscoll, Tristan P.; Caliari, Steven R.; Mehta, Kush D.; Assoian, Richard K.; Burdick, Jason A.; Mauck, Robert L.

2016-12-01

During mesenchymal development, the microenvironment gradually transitions from one that is rich in cell-cell interactions to one that is dominated by cell-ECM (extracellular matrix) interactions. Because these cues cannot readily be decoupled in vitro or in vivo, how they converge to regulate mesenchymal stem cell (MSC) mechanosensing is not fully understood. Here, we show that a hyaluronic acid hydrogel system enables, across a physiological range of ECM stiffness, the independent co-presentation of the HAVDI adhesive motif from the EC1 domain of N-cadherin and the RGD adhesive motif from fibronectin. Decoupled presentation of these cues revealed that HAVDI ligation (at constant RGD ligation) reduced the contractile state and thereby nuclear YAP/TAZ localization in MSCs, resulting in altered interpretation of ECM stiffness and subsequent changes in downstream cell proliferation and differentiation. Our findings reveal that, in an evolving developmental context, HAVDI/N-cadherin interactions can alter stem cell perception of the stiffening extracellular microenvironment.

Biophysics of cadherin adhesion.

PubMed

Leckband, Deborah; Sivasankar, Sanjeevi

2012-01-01

Since the identification of cadherins and the publication of the first crystal structures, the mechanism of cadherin adhesion, and the underlying structural basis have been studied with a number of different experimental techniques, different classical cadherin subtypes, and cadherin fragments. Earlier studies based on biophysical measurements and structure determinations resulted in seemingly contradictory findings regarding cadherin adhesion. However, recent experimental data increasingly reveal parallels between structures, solution binding data, and adhesion-based biophysical measurements that are beginning to both reconcile apparent differences and generate a more comprehensive model of cadherin-mediated cell adhesion. This chapter summarizes the functional, structural, and biophysical findings relevant to cadherin junction assembly and adhesion. We emphasize emerging parallels between findings obtained with different experimental approaches. Although none of the current models accounts for all of the available experimental and structural data, this chapter discusses possible origins of apparent discrepancies, highlights remaining gaps in current knowledge, and proposes challenges for further study.

Evidence for Fibroblast Growth Factor-2 as a Mediator of Amphetamine-Enhanced Motor Improvement following Stroke

PubMed Central

Wolf, William A.; Martin, Jody L.; Kartje, Gwendolyn L.; Farrer, Robert G.

2014-01-01

Previously we have shown that addition of amphetamine to physical therapy results in enhanced motor improvement following stroke in rats, which was associated with the formation of new motor pathways from cortical projection neurons of the contralesional cortex. It is unclear what mechanisms are involved, but amphetamine is known to induce the neuronal release of catecholamines as well as upregulate fibroblast growth factor-2 (FGF-2) expression in the brain. Since FGF-2 has been widely documented to stimulate neurite outgrowth, the present studies were undertaken to provide evidence for FGF-2 as a neurobiological mechanism underlying amphetamine-induced neuroplasticity. In the present study rats that received amphetamine plus physical therapy following permanent middle cerebral artery occlusion exhibited significantly greater motor improvement over animals receiving physical therapy alone. Amphetamine plus physical therapy also significantly increased the number of FGF-2 expressing pyramidal neurons of the contralesional cortex at 2 weeks post-stroke and resulted in significant axonal outgrowth from these neurons at 8 weeks post-stroke. Since amphetamine is a known releaser of norepinephrine, in vitro analyses focused on whether noradrenergic stimulation could lead to neurite outgrowth in a manner requiring FGF-2 activity. Primary cortical neurons did not respond to direct stimulation by norepinephrine or amphetamine with increased neurite outgrowth. However, conditioned media from astrocytes exposed to norepinephrine or isoproterenol (a beta adrenergic agonist) significantly increased neurite outgrowth when applied to neuronal cultures. Adrenergic agonists also upregulated FGF-2 expression in astrocytes. Pharmacological analysis indicated that beta receptors and alpha1, but not alpha2, receptors were involved in both effects. Antibody neutralization studies demonstrated that FGF-2 was a critical contributor to neurite outgrowth induced by astrocyte

Stability studies of extracellular domain two of neural-cadherin.

PubMed

Vunnam, Nagamani; McCool, John K; Williamson, Michael; Pedigo, Susan

2011-12-01

Neural- (NCAD) and epithelial- (ECAD) cadherin are calcium-dependent cell-adhesive molecules, and are localized at excitatory and inhibitory synapses respectively. They play an important role in synaptogenesis, synapse maintenance and plasticity. The extracellular region plays a critical role in cadherin-mediated cell adhesion, and has five tandemly repeated ectodomains (EC1-EC5). Calcium binding is required for dimer formation between first two N-terminal domains (EC1-EC2). Despite similarity in the primary structure, the extracellular domains of NCAD and ECAD have different intrinsic stability, dimerization affinity and kinetics of disassembly. To investigate the origin of these differences, we are characterizing the modular domains individually. Here, we report studies of NCAD2, EC2 of NCAD. This domain is important for calcium binding and is the physical linkage between the dimerization interface in EC1 and the membrane proximal modular domains. Thermal-denaturation studies show that NCAD2 is less stable than ECAD2 and less influenced by the adjoining 7-residue, N- and C-terminal linker segments. In addition the NCAD2 constructs are less influenced by added salt. This difference is likely due to variation in the overall number and distribution of charges on these anionic proteins. Our studies indicate that despite their sequence similarity and apparently passive role in adhesive dimer formation, EC2 of E- and N-cadherins are distinctly different and may contribute to the differences in energetics and kinetics of dimerization. Copyright © 2011 Elsevier B.V. All rights reserved.

E-cadherin: A determinant molecule associated with ovarian cancer progression, dissemination and aggressiveness

PubMed Central

Devis, Laura; Lapyckyj, Lara; Besso, María José; Llauradó, Marta; Abascal, María Florencia; Matos, María Laura; Lanau, Lucia; Castellví, Josep; Sánchez, José Luis; Pérez Benavente, Asunción; Gil-Moreno, Antonio; Reventós, Jaume; Santamaria Margalef, Anna; Rigau, Marina; Vazquez-Levin, Mónica Hebe

2017-01-01

Ovarian cancer (OC) is the fifth cancer death cause in women worldwide. The malignant nature of this disease stems from its unique dissemination pattern. Epithelial-to-mesenchymal transition (EMT) has been reported in OC and downregulation of Epithelial cadherin (E-cadherin) is a hallmark of this process. However, findings on the relationship between E-cadherin levels and OC progression, dissemination and aggressiveness are controversial. In this study, the evaluation of E-cadherin expression in an OC tissue microarray revealed its prognostic value to discriminate between advanced- and early-stage tumors, as well as serous tumors from other histologies. Moreover, E-cadherin, Neural cadherin (N-cadherin), cytokeratins and vimentin expression was assessed in TOV-112, SKOV-3, OAW-42 and OV-90 OC cell lines grown in monolayers and under anchorage-independent conditions to mimic ovarian tumor cell dissemination, and results were associated with cell aggressiveness. According to these EMT-related markers, cell lines were classified as mesenchymal (M; TOV-112), intermediate mesenchymal (IM; SKOV-3), intermediate epithelial (IE; OAW-42) and epithelial (E; OV-90). M- and IM-cells depicted the highest migration capacity when grown in monolayers, and aggregates derived from M- and IM-cell lines showed lower cell death, higher adhesion to extracellular matrices and higher invasion capacity than E- and IE-aggregates. The analysis of E-cadherin, N-cadherin, cytokeratin 19 and vimentin mRNA levels in 20 advanced-stage high-grade serous human OC ascites showed an IM phenotype in all cases, characterized by higher proportions of N- to E-cadherin and vimentin to cytokeratin 19. In particular, higher E-cadherin mRNA levels were associated with cancer antigen 125 levels more than 500 U/mL and platinum-free intervals less than 6 months. Altogether, E-cadherin expression levels were found relevant for the assessment of OC progression and aggressiveness. PMID:28934230

Cleavage and shedding of E-cadherin after induction of apoptosis.

PubMed

Steinhusen, U; Weiske, J; Badock, V; Tauber, R; Bommert, K; Huber, O

2001-02-16

Apoptotic cell death induces dramatic molecular changes in cells, becoming apparent on the structural level as membrane blebbing, condensation of the cytoplasm and nucleus, and loss of cell-cell contacts. The activation of caspases is one of the fundamental steps during programmed cell death. Here we report a detailed analysis of the fate of the Ca(2+)-dependent cell adhesion molecule E-cadherin in apoptotic epithelial cells and show that during apoptosis fragments of E-cadherin with apparent molecular masses of 24, 29, and 84 kDa are generated by two distinct proteolytic activities. In addition to a caspase-3-mediated cleavage releasing the cytoplasmic domain of E-cadherin, a metalloproteinase sheds the extracellular domain from the cell surface during apoptosis. Immunofluorescence analysis confirmed that concomitant with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulates in the cytosol. In the presence of inhibitors of caspase-3 and/or metalloproteinases, cleavage of E-cadherin was almost completely blocked. The simultaneous cleavage of the intracellular and extracellular domains of E-cadherin may provide a highly efficient mechanism to disrupt cadherin-mediated cell-cell contacts in apoptotic cells, a prerequisite for cell rounding and exit from the epithelium.

Betacellulin induces Slug-mediated down-regulation of E-cadherin and cell migration in ovarian cancer cells

PubMed Central

Zhao, Jianfang; Klausen, Christian; Qiu, Xin; Cheng, Jung-Chien; Chang, Hsun-Ming; Leung, Peter C.K.

2016-01-01

Epithelial ovarian cancer is the leading cause of death among gynaecological cancers. Previous studies have demonstrated that epidermal growth factor receptor (EGFR) ligands can induce ovarian cancer cell invasion by down-regulating E-cadherin. Betacellulin is a unique member of the EGF family. It is overexpressed in a variety of cancers and is associated with reduced survival. However, the biological functions and clinical significance of betacellulin in ovarian cancer remain unknown. In the current study, we tested the hypothesis that betacellulin induces ovarian cancer cell migration by suppressing E-cadherin expression. Treatment of SKOV3 and OVCAR5 ovarian cancer cell lines with betacellulin down-regulated E-cadherin, but not N-cadherin. In addition, betacellulin treatment increased the expression of Snail and Slug, and these effects were completely blocked by pre-treatment with EGFR inhibitor AG1478. Interestingly, only knockdown of Slug reversed the down-regulation of E-cadherin by betacellulin. Betacellulin treatment induced the activation of both the MEK-ERK and PI3K-Akt signaling pathways, and it also significantly increased ovarian cancer cell migration. Importantly, the effects of betacellulin on E-cadherin, Slug and cell migration were attenuated by pre-treatment with either U0126 or LY294002. Our results suggest that betacellulin induces ovarian cancer migration and Slug-dependent E-cadherin down-regulation via EGFR-mediated MEK-ERK and PI3K-Akt signaling. PMID:27129169

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

Changes in E-cadherin rigidity sensing regulate cell adhesion.

PubMed

Collins, Caitlin; Denisin, Aleksandra K; Pruitt, Beth L; Nelson, W James

2017-07-18

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp; Fujiwara, Yuki; Yamaguchi, Hideki

2011-05-20

Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however,more » its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.« less

Mutant Alpha-Synuclein Causes Age-Dependent Neuropathology in Monkey Brain

PubMed Central

Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua

2015-01-01

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2–3, 7–8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD. PMID:26019347

Mutant alpha-synuclein causes age-dependent neuropathology in monkey brain.

PubMed

Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua; Li, Xiao-Jiang

2015-05-27

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2-3, 7-8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD. Copyright © 2015 the authors 0270-6474/15/358345-14$15.00/0.

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).

E-cadherin junction formation involves an active kinetic nucleation process

PubMed Central

Biswas, Kabir H.; Hartman, Kevin L.; Yu, Cheng-han; Harrison, Oliver J.; Song, Hang; Smith, Adam W.; Huang, William Y. C.; Lin, Wan-Chen; Guo, Zhenhuan; Padmanabhan, Anup; Troyanovsky, Sergey M.; Dustin, Michael L.; Shapiro, Lawrence; Honig, Barry; Zaidel-Bar, Ronen; Groves, Jay T.

2015-01-01

Epithelial (E)-cadherin-mediated cell−cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role. PMID:26290581

E-cadherin junction formation involves an active kinetic nucleation process

DOE PAGES

Biswas, Kabir H.; Hartman, Kevin L.; Yu, Cheng -han; ...

2015-08-19

Epithelial (E)-cadherin-mediated cell–cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest thatmore » the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.« less

E-cadherin junction formation involves an active kinetic nucleation process

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biswas, Kabir H.; Hartman, Kevin L.; Yu, Cheng -han

Epithelial (E)-cadherin-mediated cell–cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest thatmore » the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.« less

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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Allosteric Regulation of E-Cadherin Adhesion.

PubMed

Shashikanth, Nitesh; Petrova, Yuliya I; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M; Leckband, Deborah E

2015-08-28

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Allosteric Regulation of E-Cadherin Adhesion*

PubMed Central

Shashikanth, Nitesh; Petrova, Yuliya I.; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M.; Leckband, Deborah E.

2015-01-01

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120ctn, increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120ctn dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120ctn dephosphorylation. PMID:26175155

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

PubMed Central

Larsen, Ida Signe Bohse; Narimatsu, Yoshiki; Joshi, Hiren Jitendra; Yang, Zhang; Harrison, Oliver J.; Brasch, Julia; Shapiro, Lawrence; Honig, Barry; Vakhrushev, Sergey Y.; Clausen, Henrik; Halim, Adnan

2017-01-01

Protein O-mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated α-dystroglycanopathies, because deficient O-Man glycosylation of α-dystroglycan disrupts laminin interaction with α-dystroglycan and the extracellular matrix. To explore the functions of O-Man glycans on cadherins and protocadherins, we used a combinatorial gene-editing strategy in multiple cell lines to evaluate the role of the two POMTs initiating O-Man glycosylation and the major enzyme elongating O-Man glycans, the protein O-mannose β-1,2-N-acetylglucosaminyltransferase, POMGnT1. Surprisingly, O-mannosylation of cadherins and protocadherins does not require POMT1 and/or POMT2 in contrast to α-dystroglycan, and moreover, the O-Man glycans on cadherins are not elongated. Thus, the classical and evolutionarily conserved POMT O-mannosylation pathway is essentially dedicated to α-dystroglycan and a few other proteins, whereas a novel O-mannosylation process in mammalian cells is predicted to serve the large cadherin superfamily and other proteins. PMID:28512129

The armadillo repeat region targets ARVCF to cadherin-based cellular junctions.

PubMed

Kaufmann, U; Zuppinger, C; Waibler, Z; Rudiger, M; Urbich, C; Martin, B; Jockusch, B M; Eppenberger, H; Starzinski-Powitz, A

2000-11-01

The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites. Several members of the armadillo repeat protein family mediate this linkage. We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail. Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay. Immunoprecipitation and in vitro binding assays confirmed this interaction. Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, beta-catenin and p120 (ctn). Hence, the N terminus of ARVCF is not required for junctional localization. In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes. Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.

New Fluorescent Reporter Systems for Evaluation of the Expression of E- and N-Cadherins.

PubMed

Burmistrova, O A; Nikulin, S V; Zakharova, G S; Fomicheva, K A; Alekseev, B Ya; Shkurnikov, M Yu

2018-05-24

During metastatic growth, cells of solid tumors undergo phenotypical changes related to epithelial-mesenchymal transition. Epithelial-mesenchymal transition is regarded as a potential target for prospective antitumor drugs. Fluorescent reporter systems for evaluation of the expression of markers of epithelial and mesenchymal status (E- and N-cadherins) were created. The described approaches can be used for creation of analogous reporter systems.

Charge-balanced biphasic electrical stimulation inhibits neurite extension of spiral ganglion neurons.

PubMed

Shen, Na; Liang, Qiong; Liu, Yuehong; Lai, Bin; Li, Wen; Wang, Zhengmin; Li, Shufeng

2016-06-15

Intracochlear application of exogenous or transgenic neurotrophins, such as neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF), could promote the resprouting of spiral ganglion neuron (SGN) neurites in deafened animals. These resprouting neurites might reduce the gap between cochlear implant electrodes and their targeting SGNs, allowing for an improvement of spatial resolution of electrical stimulation. This study is to investigate the impact of electrical stimulation employed in CI on the extension of resprouting SGN neurites. We established an in vitro model including the devices delivering charge-balanced biphasic electrical stimulation, and spiral ganglion (SG) dissociated culture treated with BDNF and NT-3. After electrical stimulation with varying durations and intensities, we quantified neurite lengths and Schwann cell densities in SG cultures. Stimulations that were greater than 50μA or longer than 8h significantly decreased SG neurite length. Schwann cell density under 100μA electrical stimulation for 48h was significantly lower compared to that in non-stimulated group. These electrical stimulation-induced decreases of neurite extension and Schwann cell density were attenuated by various types of voltage-dependent calcium channel (VDCC) blockers, or completely prevented by their combination, cadmium or calcium-free medium. Our study suggested that charge-balanced biphasic electrical stimulation inhibited the extension of resprouting SGN neurites and decreased Schwann cell density in vitro. Calcium influx through multiple types of VDCCs was involved in the electrical stimulation-induced inhibition. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons.

PubMed

Liu, Chuanliang; Hu, Qiongqiong; Jing, Jia; Zhang, Yun; Jin, Jing; Zhang, Liulei; Mu, Lili; Liu, Yumei; Sun, Bo; Zhang, Tongshuai; Kong, Qingfei; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Liu, Xijun; Zhao, Wei; Wang, Jinghua; Feng, Tao; Li, Hulun

2017-09-01

Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

Existence of muscarinic acetylcholine receptor (mAChR) and fibroblast growth factor receptor (FGFR) heteroreceptor complexes and their enhancement of neurite outgrowth in neural hippocampal cultures.

PubMed

Di Liberto, V; Borroto-Escuela, D O; Frinchi, M; Verdi, V; Fuxe, K; Belluardo, N; Mudò, G

2017-02-01

Recently, it was demonstrated that G-protein-coupled receptors (GPCRs) can transactivate tyrosine kinase receptors in absence of their ligands. In this work, driven by the observation that mAChRs and fibroblast growth factor receptors (FGFRs) share signalling pathways and regulation of brain functions, it was decided to explore whether mAChRs activation may transactivate FGFRs and, if so, to characterize the related trophic effects in cultured hippocampal neurons. Oxotremorine-M transactivation of FGFRs and related trophic effects were tested in primary hippocampal neurons. Western blotting and in situ proximity ligation assay (PLA) were used to detect FGFR phosphorylation (pFGFR) levels and M 1 R-FGFR1 heteroreceptor complexes, respectively. Oxotremorine-M, a non-selective mAChRs agonist, was able to transactivate FGFR and this transactivation was blocked by Src inhibitors. Oxotremorine-M treatment produced a significant increase in the primary neurite outgrowth that was blocked by pre-treatment with the pFGFR inhibitor SU5402 and Src inhibitors. This trophic effect was almost similar to that induced by fibroblast growth factor-2 (FGF-2). By using atropine as nonselective mAChRs or pirenzepine as selective antagonist for M 1 receptor (M 1 R) we could show that mAChRs are involved in modulating the pFGFRs. Using PLA, M 1 R-FGFR1 heteroreceptor complexes were identified in the hippocampus and cerebral cortex. The current findings, by showing functional mAChR-FGFR interactions, will contribute to advance the understanding of the mechanisms involved in the actions of cholinergic drugs on neuronal plasticity. Data may help to develop novel therapeutic strategies not only for neurodegenerative diseases but also for depression-induced atrophy of hippocampal neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of Cd{sup 2+} on cis-dimer structure of E-cadherin in living cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takeda, Hiroshi, E-mail: hirotake@sapmed.ac.jp

2014-02-21

Highlights: • The effects of Cd on the dimer of cadherin in living cells was analyzed. • Cd induced cadherin dimer formation was not detected in living cell with low Ca. • Ca mediated structural cooperativity and allostery in the native cadherin. • Ca concentration-dependent competitive displacement of Cd from cadherin is proposed. - Abstract: E-cadherin, a calcium (Ca{sup 2+})-dependent cell–cell adhesion molecule, plays a key role in the maintenance of tissue integrity. We have previously demonstrated that E-cadherin functions in vivo as a cis-dimer through chemical cross-linking reagents. Ca{sup 2+} plays an important role in the cis-dimer formation ofmore » cadherin. However, the molecular mechanisms by which Ca{sup 2+} interacts with the binding sites that regulate cis-dimer structures have not been completely elucidated. As expected for a Ca{sup 2+} antagonist, cadmium (Cd{sup 2+}) disrupts cadherin function by displacing Ca{sup 2+} from its binding sites on the cadherin molecules. We used Cd{sup 2+} as a probe for investigating the role of Ca{sup 2+} in the dynamics of the E-cadherin extracellular region that involve cis-dimer formation and adhesion. While cell–cell adhesion assembly was completely disrupted in the presence of Cd{sup 2+}, the amount of cis-dimers of E-cadherin that formed at the cell surface was not affected. In our “Cd{sup 2+}-switch” experiments, we did not find that Cd{sup 2+}-induced E-cadherin cis-dimer formation in EL cells when they were incubated in low-Ca{sup 2+} medium. In the present study, we demonstrated for the first time the effects of Cd{sup 2+} on the cis-dimer structure of E-cadherin in living cells using a chemical cross-link analysis.« less

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

PubMed Central

Jenkins, Jeremy L; Dean, Donald H

2001-01-01

Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research. PMID:11722800

Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in β-arrestin 2-Akt signaling and dopaminergic behaviors.

PubMed

Mototani, Yasumasa; Okamura, Tadashi; Goto, Motohito; Shimizu, Yukiko; Yanobu-Takanashi, Rieko; Ito, Aiko; Kawamura, Naoya; Yagisawa, Yuka; Umeki, Daisuke; Nariyama, Megumi; Suita, Kenji; Ohnuki, Yoshiki; Shiozawa, Kouichi; Sahara, Yoshinori; Kozasa, Tohru; Saeki, Yasutake; Okumura, Satoshi

2018-06-01

The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-β-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and β-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with β-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a β-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-β-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in β-arrestin-dependent, G protein-independent signaling.

A model for neurite growth and neuronal morphogenesis.

PubMed

Li, G H; Qin, C D

1996-02-01

A model is presented for tensile regulation of neuritic growth. It is proposed that the neurite tension can be determined by Hooke's law and determines the growth rate of neurites. The growth of a neurite is defined as the change in its unstretched length. Neuritic growth rate is assumed to increase in proportion to tension magnitude over a certain threshold [Dennerll et al., J. Cell Biol. 107: 665-674 (1988)]. The movement of branch nodes also contributes to the neuronal morphogenesis. It is supposed that the rate of a branch-node displacement is in proportion to the resultant neuritic tension exerted on this node. To deal with the growth-cone movement, it is further supposed that the environment exerts a traction force on the growth cone and the rate of growth-cone displacement is determined by the vector sum of the neuritic tension and the traction force. A group of differential equations are used to describe the model. The key point of the model is that the traction force and the neuritic tension are in opposition to generate a temporal contrast-enhancing mechanism. Results of a simulation study suggest that the model can explain some phenomena related to neuronal morphogenesis.

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

A Critical Neurodevelopmental Role for L-Type Voltage-Gated Calcium Channels in Neurite Extension and Radial Migration.

PubMed

Kamijo, Satoshi; Ishii, Yuichiro; Horigane, Shin-Ichiro; Suzuki, Kanzo; Ohkura, Masamichi; Nakai, Junichi; Fujii, Hajime; Takemoto-Kimura, Sayaka; Bito, Haruhiko

2018-06-13

Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca 2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCC during brain development remain unclear. Calcium signaling has been shown to be essential for neurodevelopmental processes such as sculpting of neurites, functional wiring, and fine tuning of growing networks. To investigate this relationship, we performed submembraneous calcium imaging using a membrane-tethered genetically encoded calcium indicator (GECI) Lck-G-CaMP7. We successfully recorded s pontaneous regenerative calcium transients (SRCaTs) in developing mouse excitatory cortical neurons prepared from both sexes before synapse formation. SRCaTs originated locally in immature neurites independently of somatic calcium rises and were significantly more elevated in the axons than in dendrites. SRCaTs were not blocked by tetrodoxin, a Na + channel blocker, but were strongly inhibited by hyperpolarization, suggesting a voltage-dependent source. Pharmacological and genetic manipulations revealed the critical importance of the Ca v 1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Ca v 1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Ca v 1.2 mutant found in Timothy syndrome, an autosomal dominant multisystem disorder exhibiting syndromic autism, resulted in impaired radial migration of layer 2/3 excitatory neurons, whereas postnatal abrogation of Ca v 1.2 enhancement could rescue cortical malformation. Together, these lines of evidence suggest a critical role for spontaneous opening of L-type VGCCs in neural development and corticogenesis and indicate that L-type VGCCs might constitute a perinatal therapeutic target for neuropsychiatric calciochannelopathies. SIGNIFICANCE STATEMENT Despite many association

Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

PubMed

Cadwell, Chantel M; Jenkins, Paul M; Bennett, Vann; Kowalczyk, Andrew P

2016-01-08

Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Mechanisms of developmental neurite pruning.

PubMed

Schuldiner, Oren; Yaron, Avraham

2015-01-01

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.

Changes in E-cadherin rigidity sensing regulate cell adhesion

PubMed Central

Collins, Caitlin; Pruitt, Beth L.; Nelson, W. James

2017-01-01

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion. PMID:28674019

Importance of ERK activation in As2O3-induced differentiation and promyelocytic leukemia nuclear bodies formation in neuroblastoma cells.

PubMed

Petit, A; Delaune, A; Falluel-Morel, A; Goullé, J-P; Vannier, J-P; Dubus, I; Vasse, M

2013-11-01

Neuroblastoma malignant cell growth is dependent on their undifferentiated status. Arsenic trioxide (As2O3) induces neuroblastoma cell differentiation in vitro, but its mechanisms still remains unknown. We used three human neuroblastoma cell lines (SH-SY5Y, IGR-N-91, LAN-1) that differ from their MYCN and p53 status to explore the intracellular events activated by As2O3 and involved in neurite outgrowth, a morphological marker of differentiation. As2O3 (2μM) induced neurite outgrowth in all cell lines, which was dependent on ERK activation but independent on MYCN status. This process was induced either by a sustained (3 days) or a transient (2h) incubation with As2O3, indicating that very early events trigger the induction of differentiation. In parallel, As2O3 induced a rapid assembly of promyelocytic leukemia nuclear bodies (PML-NB) in an ERK-dependent manner. In conclusion, mechanisms leading to neuroblastoma cell differentiation in response to As2O3 appear to involve the ERK pathway activation and PML-NB formation, which are observed in response to other differentiating molecules such as retinoic acid derivates. This open new perspectives based on the use of treatment combinations to potentiate the differentiating effects of each drug alone and reduce their adverse side effects. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structure of a force-conveying cadherin bond essential for inner-ear mechanotransduction.

PubMed

Sotomayor, Marcos; Weihofen, Wilhelm A; Gaudet, Rachelle; Corey, David P

2012-12-06

Hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals. Mechanical force from sound waves or head movements is conveyed to hair-cell transduction channels by tip links, fine filaments formed by two atypical cadherins known as protocadherin 15 and cadherin 23 (refs 4, 5). These two proteins are involved in inherited deafness and feature long extracellular domains that interact tip-to-tip in a Ca(2+)-dependent manner. However, the molecular architecture of this complex is unknown. Here we combine crystallography, molecular dynamics simulations and binding experiments to characterize the protocadherin 15-cadherin 23 bond. We find a unique cadherin interaction mechanism, in which the two most amino-terminal cadherin repeats (extracellular cadherin repeats 1 and 2) of each protein interact to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex is shown to become unstable in response to Ca(2+) removal owing to increased flexure of Ca(2+)-free cadherin repeats. Finally, we use structures and biochemical measurements to study the molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis, neural connectivity and cancer.

N-Cadherin in Prostate Cancer: Downstream Pathways and Their Translational Application for Castrate-Resistant Prostate Cancer

DTIC Science & Technology

2012-09-01

with properties of stem cells. Cell 133, 704–715 (2008). 22. Mason , M.J., Fan, G ., Plath, K., Zhou, Q. & Horvath , S. Signed weighted gene co...An J, Horvath S, Gleave M, Rettig MB, Wainberg ZA, Reiter RE. Monoclonal antibody targeting of N-cadherin inhibits prostate cancer growth, metastasis...N at u re A m er ic a, In c. A ll ri g h ts r es er ve d . A r t i c l e s nAture medicine VOLUME 16 | NUMBER 12 | DECEMBER 2010 1415 of in

Developmental neurotoxicity of different pesticides in PC-12 cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christen, Verena

The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor andmore » different concentrations of biocides for 5 days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2β, gap-43, neurofilament-h, tubulin-α and tubulin-β. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of

Rab11 in Recycling Endosomes Regulates the Sorting and Basolateral Transport of E-CadherinV⃞

PubMed Central

Lock, John G.; Stow, Jennifer L.

2005-01-01

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, ΔS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical ΔS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, μ1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion. PMID:15689490

Zinc Promotes Adipose-Derived Mesenchymal Stem Cell Proliferation and Differentiation towards a Neuronal Fate.

PubMed

Moon, Mi-Young; Kim, Hyun Jung; Choi, Bo Young; Sohn, Min; Chung, Tae Nyoung; Suh, Sang Won

2018-01-01

Zinc is an essential element required for cell division, migration, and proliferation. Under zinc-deficient conditions, proliferation and differentiation of neural progenitors are significantly impaired. Adipose-derived mesenchymal stem cells (AD-MSCs) are multipotent stem cells that can differentiate into neurons. The aim of this study was to evaluate the effect of zinc on AD-MSC proliferation and differentiation. We initially examined the effect of zinc on stem cell proliferation at the undifferentiated stage. AD-MSCs showed high proliferation rates on day 6 in 30  μ M and 100  μ M of ZnCl 2 . Zinc chelation inhibited AD-MSC proliferation via downregulation of ERK1/2 activity. We then assessed whether zinc was involved in cell migration and neurite outgrowth during differentiation. After three days of neuronal differentiation, TUJ-1-positive cells were observed, implying that AD-MSCs had differentiated into early neuron or neuron-like cells. Neurite outgrowth was increased in the zinc-treated group, while the CaEDTA-treated group showed diminished, shrunken neurites. Furthermore, we showed that zinc promoted neurite outgrowth via the inactivation of RhoA and led to the induction of neuronal gene expression (MAP2 and nestin) in differentiated stem cells. Taken together, zinc promoted AD-MSC proliferation and affected neuronal differentiation, mainly by increasing neurite outgrowth.

Zinc Promotes Adipose-Derived Mesenchymal Stem Cell Proliferation and Differentiation towards a Neuronal Fate

PubMed Central

Moon, Mi-Young; Kim, Hyun Jung; Choi, Bo Young; Sohn, Min

2018-01-01

Zinc is an essential element required for cell division, migration, and proliferation. Under zinc-deficient conditions, proliferation and differentiation of neural progenitors are significantly impaired. Adipose-derived mesenchymal stem cells (AD-MSCs) are multipotent stem cells that can differentiate into neurons. The aim of this study was to evaluate the effect of zinc on AD-MSC proliferation and differentiation. We initially examined the effect of zinc on stem cell proliferation at the undifferentiated stage. AD-MSCs showed high proliferation rates on day 6 in 30 μM and 100 μM of ZnCl2. Zinc chelation inhibited AD-MSC proliferation via downregulation of ERK1/2 activity. We then assessed whether zinc was involved in cell migration and neurite outgrowth during differentiation. After three days of neuronal differentiation, TUJ-1-positive cells were observed, implying that AD-MSCs had differentiated into early neuron or neuron-like cells. Neurite outgrowth was increased in the zinc-treated group, while the CaEDTA-treated group showed diminished, shrunken neurites. Furthermore, we showed that zinc promoted neurite outgrowth via the inactivation of RhoA and led to the induction of neuronal gene expression (MAP2 and nestin) in differentiated stem cells. Taken together, zinc promoted AD-MSC proliferation and affected neuronal differentiation, mainly by increasing neurite outgrowth. PMID:29765417

Developmental neurotoxicity of different pesticides in PC-12 cells in vitro.

PubMed

Christen, Verena; Rusconi, Manuel; Crettaz, Pierre; Fent, Karl

2017-06-15

The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2β, gap-43, neurofilament-h, tubulin-α and tubulin-β. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals

ADP-Ribosylation Factor 6 and a Functional PIX/p95-APP1 Complex Are Required for Rac1B-mediated Neurite Outgrowth

PubMed Central

Albertinazzi, Chiara; Za, Lorena; Paris, Simona; de Curtis, Ivan

2003-01-01

The mechanisms coordinating adhesion, actin organization, and membrane traffic during growth cone migration are poorly understood. Neuritogenesis and branching from retinal neurons are regulated by the Rac1B/Rac3 GTPase. We have identified a functional connection between ADP-ribosylation factor (Arf) 6 and p95-APP1 during the regulation of Rac1B-mediated neuritogenesis. P95-APP1 is an ADP-ribosylation factor GTPase-activating protein (ArfGAP) of the GIT family expressed in the developing nervous system. We show that Arf6 has a predominant role in neurite extension compared with Arf1 and Arf5. Cotransfection experiments indicate a specific and cooperative potentiation of neurite extension by Arf6 and the carboxy-terminal portion of p95-APP1. Localization studies in neurons expressing different p95-derived constructs show a codistribution of p95-APP1 with Arf6, but not Arf1. Moreover, p95-APP1–derived proteins with a mutated or deleted ArfGAP domain prevent Rac1B-induced neuritogenesis, leading to PIX-mediated accumulation at large Rab11-positive endocytic vesicles. Our data support a role of p95-APP1 as a specific regulator of Arf6 in the control of membrane trafficking during neuritogenesis. PMID:12686588

Resolving the molecular mechanism of cadherin catch bond formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manibog, Kristine; Li, Hui; Rakshit, Sabyasachi

2014-06-02

Classical cadherin Ca(2+)-dependent cell-cell adhesion proteins play key roles in embryogenesis and in maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. One of these adhesive states, called an X-dimer, forms catch bonds that strengthen and become longer lived in the presence of mechanical force. Here we use single-molecule force-clamp spectroscopy with an atomic force microscope along with molecular dynamics and steered molecular dynamics simulations to resolve the molecular mechanisms underlying catch bond formation and the role of Ca(2+) ions in this process. Our data suggest that tensile force bends the cadherin extracellular region such that theymore » form long-lived, force-induced hydrogen bonds that lock X-dimers into tighter contact. When Ca(2+) concentration is decreased, fewer de novo hydrogen bonds are formed and catch bond formation is eliminated« less

EGFR and ADAMs Cooperate to Regulate Shedding and Endocytic Trafficking of the Desmosomal Cadherin Desmoglein 2

PubMed Central

Klessner, Jodi L.; Desai, Bhushan V.; Amargo, Evangeline V.; Getsios, Spiro

2009-01-01

Regulation of classic cadherins plays a critical role in tissue remodeling during development and cancer; however, less attention has been paid to the importance of desmosomal cadherins. We previously showed that EGFR inhibition results in accumulation of the desmosomal cadherin, desmoglein 2 (Dsg2), at cell–cell interfaces accompanied by inhibition of matrix metalloprotease (MMP)-dependent shedding of the Dsg2 ectodomain and tyrosine phosphorylation of its cytoplasmic domain. Here, we show that EGFR inhibition stabilizes Dsg2 at intercellular junctions by interfering with its accumulation in an internalized cytoplasmic pool. Furthermore, MMP inhibition and ADAM17 RNAi, blocked shedding and depleted internalized Dsg2, but less so E-cadherin, in highly invasive SCC68 cells. ADAM9 and 15 silencing also impaired Dsg2 processing, supporting the idea that this desmosomal cadherin can be regulated by multiple ADAM family members. In contrast, ADAM10 siRNA enhanced accumulation of a 100-kDa Dsg2 cleavage product and internalized pool of Dsg2. Although both MMP and EGFR inhibition increased intercellular adhesive strength in control cells, the response to MMP-inhibition was Dsg2-dependent. These data support a role for endocytic trafficking in regulating desmosomal cadherin turnover and function and raise the possibility that internalization and regulation of desmosomal and classic cadherin function can be uncoupled mechanistically. PMID:18987342

Drosophila E-cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth.

PubMed

Loyer, Nicolas; Kolotuev, Irina; Pinot, Mathieu; Le Borgne, Roland

2015-10-13

Intercellular bridges called "ring canals" (RCs) resulting from incomplete cytokinesis play an essential role in intercellular communication in somatic and germinal tissues. During Drosophila oogenesis, RCs connect the maturing oocyte to nurse cells supporting its growth. Despite numerous genetic screens aimed at identifying genes involved in RC biogenesis and maturation, how RCs anchor to the plasma membrane (PM) throughout development remains unexplained. In this study, we report that the clathrin adaptor protein 1 (AP-1) complex, although dispensable for the biogenesis of RCs, is required for the maintenance of the anchorage of RCs to the PM to withstand the increased membrane tension associated with the exponential tissue growth at the onset of vitellogenesis. Here we unravel the mechanisms by which AP-1 enables the maintenance of RCs' anchoring to the PM during size expansion. We show that AP-1 regulates the localization of the intercellular adhesion molecule E-cadherin and that loss of AP-1 causes the disappearance of the E-cadherin-containing adhesive clusters surrounding the RCs. E-cadherin itself is shown to be required for the maintenance of the RCs' anchorage, a function previously unrecognized because of functional compensation by N-cadherin. Scanning block-face EM combined with transmission EM analyses reveals the presence of interdigitated, actin- and Moesin-positive, microvilli-like structures wrapping the RCs. Thus, by modulating E-cadherin trafficking, we show that the sustained E-cadherin-dependent adhesion organizes the microvilli meshwork and ensures the proper attachment of RCs to the PM, thereby counteracting the increasing membrane tension induced by exponential tissue growth.

Interaction of Neuritic Plaques and Education Predicts Dementia

PubMed Central

Roe, Catherine M.; Xiong, Chengjie; Miller, J. Phillip; Cairns, Nigel J.; Morris, John C.

2009-01-01

In exploring the cognitive reserve hypothesis in persons with substantial Alzheimer disease neuropathology, we aimed to determine the extent to which educational attainment and densities of diffuse plaques, neuritic plaques, and neurofibrillary tangles predict dementia. Participants were 1563 individuals aged 65 years or above who were assessed for dementia within 1 year of death. Generalized linear mixed models were used to examine whether education and density ratings of diffuse plaques and neuritic plaques, and neurofibrillary tangle stage were associated with a dementia diagnosis. Education interacted with densities of neuritic plaques to predict dementia. Tangle density independently predicted dementia, but did not interact with education. Diffuse plaque density was unrelated to dementia when adjusted for densities of neuritic plaques and tangles. Among individuals with Alzheimer disease neuropathology, educational attainment, as a surrogate of cognitive reserve, modifies the influence of neuritic, but not diffuse, plaque neuropathology on the expression of dementia. PMID:18525294

Drosophila E-Cadherin Functions in Hematopoietic Progenitors to Maintain Multipotency and Block Differentiation

PubMed Central

Gao, Hongjuan; Wu, Xiaorong; Fossett, Nancy

2013-01-01

A fundamental question in stem cell biology concerns the regulatory strategies that control the choice between multipotency and differentiation. Drosophila blood progenitors or prohemocytes exhibit key stem cell characteristics, including multipotency, quiescence, and niche dependence. As a result, studies of Drosophila hematopoiesis have provided important insights into the molecular mechanisms that control these processes. Here, we show that E-cadherin is an important regulator of prohemocyte fate choice, maintaining prohemocyte multipotency and blocking differentiation. These functions are reminiscent of the role of E-cadherin in mammalian embryonic stem cells. We also show that mis-expression of E-cadherin in differentiating hemocytes disrupts the boundary between these cells and undifferentiated prohemocytes. Additionally, upregulation of E-cadherin in differentiating hemocytes increases the number of intermediate cell types expressing the prohemocyte marker, Patched. Furthermore, our studies indicate that the Drosophila GATA transcriptional co-factor, U-shaped, is required for E-cadherin expression. Consequently, E-cadherin is a downstream target of U-shaped in the maintenance of prohemocyte multipotency. In contrast, we showed that forced expression of the U-shaped GATA-binding partner, Serpent, repressed E-cadherin expression and promoted lamellocyte differentiation. Thus, U-shaped may maintain E-cadherin expression by blocking the inhibitory activity of Serpent. Collectively, these observations suggest that GATA:FOG complex formation regulates E-cadherin levels and, thereby, the choice between multipotency and differentiation. The work presented in this report further defines the molecular basis of prohemocyte cell fate choice, which will provide important insights into the mechanisms that govern stem cell biology. PMID:24040319

Cadherin-2 Is Required Cell Autonomously for Collective Migration of Facial Branchiomotor Neurons.

PubMed

Rebman, Jane K; Kirchoff, Kathryn E; Walsh, Gregory S

2016-01-01

Collective migration depends on cell-cell interactions between neighbors that contribute to their overall directionality, yet the mechanisms that control the coordinated migration of neurons remains to be elucidated. During hindbrain development, facial branchiomotor neurons (FBMNs) undergo a stereotypic tangential caudal migration from their place of birth in rhombomere (r)4 to their final location in r6/7. FBMNs engage in collective cell migration that depends on neuron-to-neuron interactions to facilitate caudal directionality. Here, we demonstrate that Cadherin-2-mediated neuron-to-neuron adhesion is necessary for directional and collective migration of FBMNs. We generated stable transgenic zebrafish expressing dominant-negative Cadherin-2 (Cdh2ΔEC) driven by the islet1 promoter. Cell-autonomous inactivation of Cadherin-2 function led to non-directional migration of FBMNs and a defect in caudal tangential migration. Additionally, mosaic analysis revealed that Cdh2ΔEC-expressing FBMNs are not influenced to migrate caudally by neighboring wild-type FBMNs due to a defect in collective cell migration. Taken together, our data suggest that Cadherin-2 plays an essential cell-autonomous role in mediating the collective migration of FBMNs.

Inhibition of the kynurenine pathway protects against reactive microglial-associated reductions in the complexity of primary cortical neurons.

PubMed

O'Farrell, Katherine; Fagan, Eimear; Connor, Thomas J; Harkin, Andrew

2017-09-05

Brain glia possess the rate limiting enzyme indoleamine 2, 3-dioxygenase (IDO) which catalyses the conversion of tryptophan to kynurenine. Microglia also express kynurenine monooxygenase (KMO) and kynureninase (KYNU) which lead to the production of the free radical producing metabolites, 3-hydroxykynurenine and 3-hydroxyanthranillic acid respectively and subsequently production of the NMDA receptor agonist quinolinic acid. The aim of this study was to examine the effect of IFNγ-stimulated kynurenine pathway (KP) induction in microglia on neurite outgrowth and complexity, and to determine whether alterations could be abrogated using pharmacological inhibitors of the KP. BV-2 microglia were treated with IFNγ (5ng/ml) for 24h and conditioned media (CM) was placed on primary cortical neurons 3 days in vitro (DIV) for 48h. Neurons were fixed and neurite outgrowth and complexity was assessed using fluorescent immunocytochemistry followed by Sholl analysis. Results show increased mRNA expression of IDO, KMO and KYNU, and increased concentrations of tryptophan, kynurenine, and 3-hydroxykynurenine in the CM of IFNγ-stimulated BV-2 microglia. The IFNγ-stimulated BV-2 microglial CM reduced neurite outgrowth and complexity with reductions in various parameters of neurite outgrowth prevented when BV-2 microglia were pre-treated with either the IDO inhibitor, 1-methyltryptophan (1-MT) (L) (0.5mM; 30min), the KMO inhibitor, Ro 61-8048 (1μM; 30min), the synthetic glucocorticoid, dexamethasone (1μM; 2h) -which suppresses IFNγ-induced IDO - and the N-methyl-D-aspartate (NMDA) receptor antagonist, MK801 (0.1μM; 30min). Overall this study indicates that inhibition of the KP in microglia may be targeted to protect against reactive microglial-associated neuronal atrophy. Copyright © 2017 Elsevier B.V. All rights reserved.

Suppression of proliferation and neurite extension of human neuroblastoma SH-SY5Y cells on immobilized Psathyrella velutina lectin.

PubMed

Kitamura, Noriaki; Ikekita, Masahiko; Hayakawa, Satoru; Funahashi, Hisayuki; Furukawa, Kiyoshi

2004-02-01

Glycoproteins from mammalian brain tissues contain unique N-linked oligosaccharides terminating with beta-N-acetylglucosamine residues. Lectin blot analysis of membrane glycoprotein samples from human neuroblastoma SH-SY5Y cells showed that several protein bands bind to Psathylera velutina lectin (PVL), which interacts with beta-N-acetylglucosamine-terminating oligosaccharides. No lectin positive bands were detected by digestion with jack bean beta-N-acetyl-hexosaminidase or N-glycanase before incubation with the lectin, indicating that the cells contain beta-N-acetylglucosamine-terminating N-linked oligosaccharides. When cells were cultured in dishes with different concentrations of PVL, the cell proliferation was inhibited in a dose-dependent manner. Similarly, the neurite extension, which was stimulated with nerve growth factor, was also inhibited in a manner dependent on the lectin dose. Cell proliferation and neurite extension were recovered by the addition of 10 mM N-acetylglucosamine into the medium. Immunoblot analysis of the activation of mitogen-activated protein (MAP) kinases and protein kinase C revealed that phosphorylation of 42-kDa and 44-kDa MAP kinases and 80-kDa protein kinase C are inhibited when SH-SY5Y cells are cultured in PVL-coated dishes, but are restored by the addition of the haptenic sugar into the medium, indicating that MAP kinase and protein kinase C pathways are inhibited by interaction with immobilized PVL. These results indicate that beta-N-acetylglucosamine-terminating N-linked oligosaccharides expressed on neural cells can induce intracellular signals upon binding to extracellular receptors, and are important for growth regulation of neural cells. Copyright 2003 Wiley-Liss, Inc.

DNA methylation-induced E-cadherin silencing is correlated with the clinicopathological features of melanoma.

PubMed

Venza, Mario; Visalli, Maria; Catalano, Teresa; Biondo, Carmelo; Beninati, Concetta; Teti, Diana; Venza, Isabella

2016-04-01

E-cadherin, a calcium-dependent cell-cell adhesion molecule, has an important role in epithelial cell function, maintenance of tissue architecture and cancer suppression. Loss of E-cadherin promotes tumor metastatic dissemination and predicts poor prognosis. The present study investigated the clinicopathological significance of E-cadherin expression in cutaneous, mucosal and uveal melanoma related to epigenetic mechanisms that may contribute to E-cadherin silencing. E-cadherin expression was reduced in 55/130 cutaneous (42.3%), 49/82 mucosal (59.7%) and 36/64 uveal (56.2%) melanoma samples as compared to normal skin controls and was inversely associated with promoter methylation. Of the 10 different CpG sites studied (nt 863, 865, 873, 879, 887, 892, 901, 918, 920 and 940), two sites (nt 892 and 940) were 90-100% methylated in all the melanoma specimens examined and the other ones were partially methylated (range, 53-86%). In contrast, the methylation rate of the E-cadherin gene was low in normal tissues (range, 5-24%). In all the three types of melanoma studied, a significant correlation was found between reduced levels of E-cadherin and reduced survival, high mitotic index and metastasis, accounting for the predilection of lymph nodal localization. In cutaneous and mucosal melanoma, low E-cadherin expression was positively correlated also with head/neck localization and ulceration. A high frequency of reduced E-cadherin levels occurred in choroid melanomas. In vitro experiments showed that E-cadherin transcription was restored following 5-aza-2'-deoxycytidine (5-aza-dC) treatment or DNMT1 silencing and was negatively correlated with the invasive potential of melanoma cells. The significant relationship between E-cadherin silencing and several poor prognostic factors indicates that this adhesion molecule may play an important role in melanomagenesis. Therefore, the inverse association of E-cadherin expression with promoter methylation raises the intriguing

cAMP-responsive Element-binding Protein (CREB) and cAMP Co-regulate Activator Protein 1 (AP1)-dependent Regeneration-associated Gene Expression and Neurite Growth*

PubMed Central

Ma, Thong C.; Barco, Angel; Ratan, Rajiv R.; Willis, Dianna E.

2014-01-01

To regenerate damaged axons, neurons must express a cassette of regeneration-associated genes (RAGs) that increases intrinsic growth capacity and confers resistance to extrinsic inhibitory cues. Here we show that dibutyrl-cAMP or forskolin combined with constitutive-active CREB are superior to either agent alone in driving neurite growth on permissive and inhibitory substrates. Of the RAGs examined, only arginase 1 (Arg1) expression correlated with the increased neurite growth induced by the cAMP/CREB combination, both of which were AP1-dependent. This suggests that cAMP-induced AP1 activity is necessary and interacts with CREB to drive expression of RAGs relevant for regeneration and demonstrates that combining a small molecule (cAMP) with an activated transcription factor (CREB) stimulates the gene expression necessary to enhance axonal regeneration. PMID:25296755

Cadherin-11 modulates cell morphology and collagen synthesis in periodontal ligament cells under mechanical stress.

PubMed

Feng, Lishu; Zhang, Yimei; Kou, Xiaoxing; Yang, Ruili; Liu, Dawei; Wang, Xuedong; Song, Yang; Cao, Haifeng; He, Danqing; Gan, Yehua; Zhou, Yanheng

2017-03-01

To examine the role of cadherin-11, an integral membrane adhesion molecule, in periodontal ligament cells (PDLCs) under mechanical stimulation. Human PDLCs were cultured and subjected to mechanical stress. Cadherin-11 expression and cell morphology of PDLCs were investigated via immunofluorescence staining. The mRNA and protein expressions of cadherin-11 and type I collagen (Col-I) of PDLCs were evaluated by quantitative real-time polymerase chain reaction and Western blot, respectively. Small interfering RNA was used to knock down cadherin-11 expression in PDLCs. The collagen matrix of PDLCs was examined using toluidine blue staining. Cadherin-11 was expressed in PDLCs. Mechanical stress suppressed cadherin-11 expression in PDLCs with prolonged force treatment time and increased force intensity, accompanied by suppressed β-catenin expression. Simultaneously, mechanical stress altered cell morphology and repressed Col-I expression in a time- and dose-dependent manner in PDLCs. Moreover, knockdown of cadherin-11 with suppressed β-catenin expression resulted in altered PDLC morphology and repressed collagen expression, which were consistent with the changes observed under mechanical stress. Results of this study suggest that cadherin-11 is expressed in PDLCs and modulates PDLC morphology and collagen synthesis in response to mechanical stress, which may play an important role in the homeostasis and remodeling of the PDL under mechanical stimulation.

Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent

PubMed Central

Carter, Emma J.; Barnes, David; Hoppe, Hans-Jürgen; Hughes, Jennifer; Cobbold, Stephen; Harper, James; Morreau, Hans; Surakhy, Mirvat; Hassan, A. Bassim

2016-01-01

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear β-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and β-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits β-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity. PMID:27566565

Surface engineered magnetic nanoparticles for specific immunotargeting of cadherin expressing cells

NASA Astrophysics Data System (ADS)

Moros, Maria; Delhaes, Flavien; Puertas, Sara; Saez, Berta; de la Fuente, Jesús M.; Grazú, Valeria; Feracci, Helene

2016-02-01

In spite of historic advances in cancer biology and recent development of sophisticated chemotherapeutics, the outlook for patients with advanced cancer is still grim. In this sense nanoparticles (NPs), through their unique physical properties, enable the development of new approaches for cancer diagnosis and treatment. Thus far the most used active targeting scheme involves NPs functionalization with antibodies specific to molecules overexpressed on cancer cell’s surface. Therefore, such active targeting relies on differences in NPs uptake kinetics rates between tumor and healthy cells. Many cancers of epithelial origin are associated with the inappropriate expression of non-epithelial cadherins (e.g. N-, P-, -11) with concomitant loss of E-cadherin. Such phenomenon named cadherin switching favors tumor development and metastasis via interactions of tumor cells with stromal components. That is why we optimized the oriented functionalization of fluorescently labelled magnetic NPs with a novel antibody specific for the extracellular domain of cadherin-11. The obtained Ab-NPs exhibited high specificity when incubated with two cell lines used as models of tumor and healthy cells. Thus, cadherin switching offers a great opportunity for the development of active targeting strategies aimed to improve the early detection and treatment of cancer.

Slug, Twist, and E-Cadherin as Immunohistochemical Biomarkers in Meningeal Tumors

PubMed Central

Nagaishi, Masaya; Nobusawa, Sumihito; Tanaka, Yuko; Ikota, Hayato; Yokoo, Hideaki; Nakazato, Yoichi

2012-01-01

The overexpression of Twist and Slug and subsequent down-regulation of E-cadherin facilitate the acquirement of invasive growth properties in cancer cells. It is unclear which of these molecules are expressed in mesenchymal tumors in the central nervous system. Here, we investigated 10 cases each of hemangiopericytoma, solitary fibrous tumor, meningothelial, fibrous, angiomatous, and atypical meningiomas, and 5 cases of anaplastic meningioma for Slug, Twist, E-cadherin, and N-cadherin immunoexpression. Nuclear Slug expression was observed in 9/10 (90%) hemangiopericytomas and 5/10 (50%) solitary fibrous tumors, but not in any meningiomas, except for 1 case. Similarly, nuclear Twist expression was more extensive in hemangiopericytomas and solitary fibrous tumors than meningiomas. In contrast to Slug and Twist, the positive expression of E-cadherin was observed in 39/45 (87%) meningiomas, but not in any hemangiopericytomas or solitary fibrous tumors (P<0.0001). The fraction of tumor cells expressing E-cadherin in meningeal tumors was negatively correlated to those of Twist (P = 0.004) and Slug (P<0.0001). The overexpression of Slug and Twist with down-regulation of E-cadherin was characteristic findings in hemangiopericytomas and solitary fibrous tumors, but not in meningiomas. The immunohistochemical profiles of the two tumor groups may be useful as diagnostic markers in cases that present a differential diagnosis challenge. PMID:23029385

Association of N-cadherin levels and downstream effectors of Rho GTPases with dendritic spine loss induced by chronic stress in rat hippocampal neurons.

PubMed

Castañeda, Patricia; Muñoz, Mauricio; García-Rojo, Gonzalo; Ulloa, José L; Bravo, Javier A; Márquez, Ruth; García-Pérez, M Alexandra; Arancibia, Damaris; Araneda, Karina; Rojas, Paulina S; Mondaca-Ruff, David; Díaz-Véliz, Gabriela; Mora, Sergio; Aliaga, Esteban; Fiedler, Jenny L

2015-10-01

Chronic stress promotes cognitive impairment and dendritic spine loss in hippocampal neurons. In this animal model of depression, spine loss probably involves a weakening of the interaction between pre- and postsynaptic cell adhesion molecules, such as N-cadherin, followed by disruption of the cytoskeleton. N-cadherin, in concert with catenin, stabilizes the cytoskeleton through Rho-family GTPases. Via their effector LIM kinase (LIMK), RhoA and ras-related C3 botulinum toxin substrate 1 (RAC) GTPases phosphorylate and inhibit cofilin, an actin-depolymerizing molecule, favoring spine growth. Additionally, RhoA, through Rho kinase (ROCK), inactivates myosin phosphatase through phosphorylation of the myosin-binding subunit (MYPT1), producing actomyosin contraction and probable spine loss. Some micro-RNAs negatively control the translation of specific mRNAs involved in Rho GTPase signaling. For example, miR-138 indirectly activates RhoA, and miR-134 reduces LIMK1 levels, resulting in spine shrinkage; in contrast, miR-132 activates RAC1, promoting spine formation. We evaluated whether N-cadherin/β-catenin and Rho signaling is sensitive to chronic restraint stress. Stressed rats exhibit anhedonia, impaired associative learning, and immobility in the forced swim test and reduction in N-cadherin levels but not β-catenin in the hippocampus. We observed a reduction in spine number in the apical dendrites of CA1 pyramidal neurons, with no effect on the levels of miR-132 or miR-134. Although the stress did not modify the RAC-LIMK-cofilin signaling pathway, we observed increased phospho-MYPT1 levels, probably mediated by RhoA-ROCK activation. Furthermore, chronic stress raises the levels of miR-138 in accordance with the observed activation of the RhoA-ROCK pathway. Our findings suggest that a dysregulation of RhoA-ROCK activity by chronic stress could potentially underlie spine loss in hippocampal neurons. © 2015 Wiley Periodicals, Inc.

Low-expression of E-cadherin in leukaemia cells causes loss of homophilic adhesion and promotes cell growth.

PubMed

Rao, Qing; Wang, Ji-Ying; Meng, Jihong; Tang, Kejing; Wang, Yanzhong; Wang, Min; Xing, Haiyan; Tian, Zheng; Wang, Jianxiang

2011-09-01

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.

Peripheral Nerve Repair in Rats Using Composite Hydrogel-Filled Aligned Nanofiber Conduits with Incorporated Nerve Growth Factor

PubMed Central

Jin, Jenny; Limburg, Sonja; Joshi, Sunil K.; Landman, Rebeccah; Park, Michelle; Zhang, Qia; Kim, Hubert T.

2013-01-01

Repair of peripheral nerve defects with current synthetic, tubular nerve conduits generally shows inferior recovery when compared with using nerve autografts, the current gold standard. We tested the ability of composite collagen and hyaluronan hydrogels, with and without the nerve growth factor (NGF), to stimulate neurite extension on a promising aligned, nanofiber poly-L-lactide-co-caprolactone (PLCL) scaffold. In vitro, the hydrogels significantly increased neurite extension from dorsal root ganglia explants. Consistent with these results, the addition of hydrogels as luminal fillers within aligned, nanofiber tubular PLCL conduits led to improved sensory function compared to autograft repair in a critical-size defect in the sciatic nerve in a rat model. Sensory recovery was assessed 3 and 12 weeks after repair using a withdrawal assay from thermal stimulation. The addition of hydrogel did not enhance recovery of motor function in the rat model. The NGF led to dose-dependent improvements in neurite out-growth in vitro, but did not have a significant effect in vivo. In summary, composite collagen/hyaluronan hydrogels enhanced sensory neurite outgrowth in vitro and sensory recovery in vivo. The use of such hydrogels as luminal fillers for tubular nerve conduits may therefore be useful in assisting restoration of protective sensation following peripheral nerve injury. PMID:23659607

Cell density and N-cadherin interactions regulate cell proliferation in the sensory epithelia of the inner ear.

PubMed

Warchol, Mark E

2002-04-01

Sensory hair cells in the inner ears of nonmammalian vertebrates can regenerate after injury. In many species, replacement hair cells are produced by the proliferation of epithelial supporting cells. Thus, the ability of supporting cells to undergo renewed proliferation is a key determinant of regenerative ability. The present study used cultures of isolated inner ear sensory epithelia to identify cellular signals that regulate supporting cell proliferation. Small pieces of sensory epithelia from the chicken utricle were cultured in glass microwells. Under those conditions, cell proliferation was inversely related to local cell density. The signaling molecules N-cadherin, beta-catenin, and focal adhesion kinase were immunolocalized in the cultured epithelial cells, and high levels of phosphotyrosine immunoreactivity were present at cell-cell junctions and focal contacts of proliferating cells. Binding of microbeads coated with a function-blocking antibody to N-cadherin inhibited ongoing proliferation. The growth of epithelial cells was also affected by the density of extracellular matrix molecules. The results suggest that cell density, cell-cell contact, and the composition of the extracellular matrix may be critical influences on the regulation of sensory regeneration in the inner ear.

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

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

PubMed

Rezaei, Maryam; Cao, Jiahui; Friedrich, Katrin; Kemper, Björn; Brendel, Oliver; Grosser, Marianne; Adrian, Manuela; Baretton, Gustavo; Breier, Georg; Schnittler, Hans-Joachim

2018-01-01

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased β-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

Promotion of neurite and filopodium formation by CD47: roles of integrins, Rac, and Cdc42.

PubMed

Miyashita, Motoaki; Ohnishi, Hiroshi; Okazawa, Hideki; Tomonaga, Hiroyasu; Hayashi, Akiko; Fujimoto, Tetsuro-Takahiro; Furuya, Nobuhiko; Matozaki, Takashi

2004-08-01

Axon extension during development is guided by many factors, but the signaling mechanisms responsible for its regulation remain largely unknown. We have now investigated the role of the transmembrane protein CD47 in this process in N1E-115 neuroblastoma cells. Forced expression of CD47 induced the formation of neurites and filopodia. Furthermore, an Fc fusion protein containing the extracellular region of the CD47 ligand SHPS-1 induced filopodium formation, and this effect was enhanced by CD47 overexpression. SHPS-1-Fc also promoted neurite and filopodium formation triggered by serum deprivation. Inhibition of Rac or Cdc42 preferentially blocked CD47-induced formation of neurites and filopodia, respectively. Overexpression of CD47 resulted in the activation of both Rac and Cdc42. The extracellular region of CD47 was sufficient for the induction of neurite formation by forced expression, but the entire structure of CD47 was required for enhancement of filopodium formation by SHPS-1-Fc. Neurite formation induced by CD47 was also inhibited by a mAb to the integrin beta3 subunit. These results indicate that the interaction of SHPS-1 with CD47 promotes neurite and filopodium formation through the activation of Rac and Cdc42, and that integrins containing the beta3 subunit participate in the effect of CD47 on neurite formation.

The indole compound NC009-1 inhibits aggregation and promotes neurite outgrowth through enhancement of HSPB1 in SCA17 cells and ameliorates the behavioral deficits in SCA17 mice.

PubMed

Chen, Chiung-Mei; Chen, Wan-Ling; Hung, Chen-Ting; Lin, Te-Hsien; Chao, Chih-Ying; Lin, Chih-Hsin; Wu, Yih-Ru; Chang, Kuo-Hsuan; Yao, Ching-Fa; Lee-Chen, Guey-Jen; Su, Ming-Tsan; Hsieh-Li, Hsiu Mei

2018-06-21

Spinocerebellar ataxia type 17 (SCA17) is caused by the expansion of translated CAG repeat in the TATA box binding protein (TBP) gene encoding a long polyglutamine (polyQ) tract in the TBP protein, which leads to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On cells with inducible SCA17 TBP/Q 79 -GFP expression to test five in-house NC009 indole compounds for neuroprotection. We found that both aggregation and polyQ-induced reactive oxygen species can be significantly prohibited by the tested NC009 compounds in Tet-On TBP/Q 79 293 cells. Among the five indole compounds, NC009-1 up-regulated expression of heat shock protein family B (small) member 1 (HSPB1) chaperone to reduce polyQ aggregation and promote neurite outgrowth in neuronal differentiated TBP/Q 79 SH-SY5Y cells. The increased HSPB1 thus ameliorated the increased BH3 interacting domain death agonist (BID), cytochrome c (CYCS) release, and caspase 3 (CASP3) activation which result in apoptosis. Knock down of HSPB1 attenuated the effects of NC009-1 on TBP/Q 79 SH-SY5Y cells, suggesting that HSPB1 might be one of the major pathways involved for NC009-1 effects. NC009-1 further reduced polyQ aggregation in Purkinje cells and ameliorated behavioral deficits in SCA17 TBP/Q 109 transgenic mice. Our results suggest that NC009-1 has a neuroprotective effect on SCA17 cell and mouse models to support its therapeutic potential in SCA17 treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

E-cadherin in contact inhibition and cancer.

PubMed

Mendonsa, Alisha M; Na, Tae-Young; Gumbiner, Barry M

2018-05-21

E-cadherin is a key component of the adherens junctions that are integral in cell adhesion and maintaining epithelial phenotype of cells. Homophilic E-cadherin binding between cells is important in mediating contact inhibition of proliferation when cells reach confluence. Loss of E-cadherin expression results in loss of contact inhibition and is associated with increased cell motility and advanced stages of cancer. In this review we discuss the role of E-cadherin and its downstream signaling in regulation of contact inhibition and the development and progression of cancer.

Experimental microembolism induces localized neuritic pathology in guinea pig cerebrum

PubMed Central

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

2015-01-01

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

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.

E-cadherin regulators are differentially expressed in the epithelium and stroma of keratocystic odontogenic tumors.

PubMed

Porto, Lia Pontes Arruda; dos Santos, Jean Nunes; Ramalho, Luciana Maria Pedreira; Figueiredo, Andreia Leal; Carneiro Júnior, Bráulio; Gurgel, Clarissa Araújo; Paiva, Katiúcia Batista Silva; Xavier, Flávia Caló Aquino

2016-04-01

The epithelial-mesenchymal transition (EMT) is the process where cells lose their epithelial features and acquire properties of typical mesenchymal cells. The dissociation of tumor cells due to changes in cell-cell adhesion is one of the key principles of tumor invasion and EMT. Thus, the knowledge of the molecular features of EMT in keratocyst odontogenic tumor (KOT) can provide useful markers to aid in the diagnosis and prognosis and perhaps contribute to an alternative therapeutic approach as it shows an aggressive clinical behavior and high recurrence rates. This study aimed to evaluate the EMT in KOT by the immunoexpression of E-cadherin, N-cadherin, Snail, and Slug and comparing to radicular cysts and dental follicles. Thirty-two KOTs, 15 radicular cysts, and 08 dental follicles were used for immunohistochemistry, evaluating the extent, intensity, labeling pattern, cellular compartment in the epithelium and stroma, and the presence of inflammation. E-cadherin was preserved in most cases of keratocystic odontogenic tumor. N-cadherin was increased in the tumor epithelium, a result that was positively correlated with the heterogeneous and nuclear immunoexpression of Slug in the epithelium; Slug also correlated with high Snail immunoexpression. N-cadherin was positively correlated with Slug in the stroma of keratocystic odontogenic tumors. The high immunoexpression of Snail and nuclear Slug in keratocystic odontogenic tumors suggests these proteins as transcription factors without necessarily participating in 'cadherin switching'. However, the knowledge of their induction of the epithelial-mesenchymal transition in odontogenic tumors is still limited. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Matrix metalloproteinase-2 is downregulated in sciatic nerve by streptozotocin induced diabetes and/or treatment with minocycline: Implications for nerve regeneration

PubMed Central

Ali, Sumia; Driscoll, Heather E.; Newton, Victoria L.; Gardiner, Natalie J.

2014-01-01

Minocycline is an inhibitor of matrix metalloproteinases (MMPs) and has been shown to have analgesic effects. Whilst increased expression of MMPs is associated with neuropathic pain, MMPs also play crucial roles in Wallerian degeneration and nerve regeneration. In this study we examined the expression of MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1/-2 in the sciatic nerve of control and streptozotocin-induced diabetic rats treated with either vehicle or minocycline by quantitative PCR and gelatin zymography. We assessed the effects of minocycline on nerve conduction velocity and intraepidermal nerve fibre (IENF) deficits in diabetic neuropathy and investigated the effects of minocycline or MMP-2 on neurite outgrowth from primary cultures of dissociated adult rat sensory neurons. We show that MMP-2 is expressed constitutively in the sciatic nerve in vivo and treatment with minocycline or diabetes leads to downregulation of MMP-2 expression and activity. The functional consequence of this is IENF deficits in minocycline-treated nondiabetic rats and an unsupportive microenvironment for regeneration in diabetes. Minocycline reduces levels of MMP-2 mRNA and nerve growth factor-induced neurite outgrowth. Furthermore, in vivo minocycline treatment reduces preconditioning-induced in vitro neurite outgrowth following a sciatic nerve crush. In contrast, the addition of active MMP-2 facilitates neurite outgrowth in the absence of neurotrophic support and pre-treatment of diabetic sciatic nerve substrata with active MMP-2 promotes a permissive environment for neurite outgrowth. In conclusion we suggest that MMP-2 downregulation may contribute to the regenerative deficits in diabetes. Minocycline treatment also downregulates MMP-2 activity and is associated with inhibitory effects on sensory neurons. Thus, caution should be exhibited with its use as the balance between beneficial and detrimental outcomes may be critical in assessing the benefits of using

Plasticity Related Gene 3 (PRG3) overcomes myelin-associated growth inhibition and promotes functional recovery after spinal cord injury

PubMed Central

Broggini, Thomas; Schnell, Lisa; Ghoochani, Ali; Mateos, José María; Buchfelder, Michael; Wiendieck, Kurt; Schäfer, Michael K.; Eyupoglu, Ilker Y.; Savaskan, Nicolai E.

2016-01-01

The Plasticity Related Gene family covers five, brain-specific, transmembrane proteins (PRG1-5, also termed LPPR1-5) that operate in neuronal plasticity during development, aging and brain trauma. Here we investigated the role of the PRG family on axonal and filopodia outgrowth. Comparative analysis revealed the strongest outgrowth induced by PRG3 (LPPR1). During development, PRG3 is ubiquitously located at the tip of neuronal processes and at the plasma membrane and declines with age. In utero electroporation of PRG3 induced dendritic protrusions and accelerated spine formations in cortical pyramidal neurons. The neurite growth promoting activity of PRG3 requires RasGRF1 (RasGEF1/Cdc25) mediated downstream signaling. Moreover, in axon collapse assays, PRG3-induced neurites resisted growth inhibitors such as myelin, Nogo-A (Reticulon/RTN-4), thrombin and LPA and impeded the RhoA-Rock-PIP5K induced neurite repulsion. Transgenic adult mice with constitutive PRG3 expression displayed strong axonal sprouting distal to a spinal cord lesion. Moreover, fostered PRG3 expression promoted complex motor-behavioral recovery compared to wild type controls as revealed in the Schnell swim test (SST). Thus, PRG3 emerges as a developmental RasGRF1-dependent conductor of filopodia formation and axonal growth enhancer. PRG3-induced neurites resist brain injury-associated outgrowth inhibitors and contribute to functional recovery after spinal cord lesions. Here, we provide evidence that PRG3 operates as an essential neuronal growth promoter in the nervous system. Maintaining PRG3 expression in aging brain may turn back the developmental clock for neuronal regeneration and plasticity. PMID:27744421

Influence of Different ECM-Like Hydrogels on Neurite Outgrowth Induced by Adipose Tissue-Derived Stem Cells

PubMed Central

Oliveira, E.; Assunção-Silva, R. C.; Teixeira, F. G.

2017-01-01

Mesenchymal stem cells (MSCs) have been proposed for spinal cord injury (SCI) applications due to their capacity to secrete growth factors and vesicles—secretome—that impacts important phenomena in SCI regeneration. To improve MSC survival into SCI sites, hydrogels have been used as transplantation vehicles. Herein, we hypothesized if different hydrogels could interact differently with adipose tissue-derived MSCs (ASCs). The efficacy of three natural hydrogels, gellan gum (functionalized with a fibronectin peptide), collagen, and a hydrogel rich in laminin epitopes (NVR-gel) in promoting neuritogenesis (alone and cocultured with ASCs), was evaluated in the present study. Their impact on ASC survival, metabolic activity, and gene expression was also evaluated. Our results indicated that all hydrogels supported ASC survival and viability, being this more evident for the functionalized GG hydrogels. Moreover, the presence of different ECM-derived biological cues within the hydrogels appears to differently affect the mRNA levels of growth factors involved in neuronal survival, differentiation, and axonal outgrowth. All the hydrogel-based systems supported axonal growth mediated by ASCs, but this effect was more robust in functionalized GG. The data herein presented highlights the importance of biological cues within hydrogel-based biomaterials as possible modulators of ASC secretome and its effects for SCI applications. PMID:29333166

Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin

PubMed Central

Broders-Bondon, Florence; Paul-Gilloteaux, Perrine; Gazquez, Elodie; Heysch, Julie; Piel, Matthieu; Mayor, Roberto; Lambris, John D.; Dufour, Sylvie

2016-01-01

We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cad-herin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cad-herin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders. PMID:27041467

Opposite Roles of Furin and PC5A in N-Cadherin Processing12