Recombinant myelin-associated glycoprotein confers neural adhesion and neurite outgrowth function

Myelin-associated glycoprotein (MAG) cDNA clones for the small (p67) and large (p72) forms were expressed in heterologous cells. Purified recombinant MAG protein was incorporated into fluorescent liposomes, and both forms were shown to bind predominantly to neurites in DRG or spinal cord cultures. This adhesion was completely blocked by Fab fragments of monoclonal anti-MAG antibody. Liposomes prepared with the control protein glycophorin or no protein failed to bind neurites. Small cerebellar neurons, which are not myelinated in vivo, failed to bind MAG liposomes. In a second test of function, p67 MAG-transfected fibroblasts were markedly enhanced in their ability to promote DRG neurite extension over a 2 day culture period compared with control fibroblasts not expressing MAG. Neurite extension was blocked by anti-MAG antibodies. These results show that both forms of MAG can facilitate the interactions between glial cells and neurites that ultimately lead to myelin formation.     

Ganglioside modulation of neural cell adhesion molecule and N-cadherin- dependent neurite outgrowth

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human neural cell adhesion molecule (NCAM) or chick N-cadherin as a culture substrate for PC12 cells. NCAM and N-cadherin in the monolayer directly promote neurite outgrowth from PC12 cells via a G-protein-dependent activation of neuronal calcium channels. In the present study we show that ganglioside GM1 does not directly activate this pathway in PC12 cells. However, the presence of GM1 (12.5-100 micrograms/ml) in the co-culture was associated with a potentiation of NCAM and N-cadherin-dependent neurite outgrowth. Treatment of PC12 cells with GM1 (100 micrograms/ml) for 90 min led to trypsin-stable increases in both beta-cholera toxin binding to PC12 cells and an enhanced neurite outgrowth response to N-cadherin. The ganglioside response could be fully inhibited by treatment with pertussis toxin. These data are consistent with exogenous gangliosides enhancing neuritic growth by promoting cell adhesion molecule-induced calcium influx into neurons.     

Sensitivity of neurite outgrowth to microfilament disruption varies with adhesion molecule substrate

Interactions between the cytoskeleton and cell adhesion molecules are presumed responsible for neurite extension. We have examined the role of microfilaments in neurite outgrowth on the cell adhesion molecules L1, P84, N-CAM, and on laminin. Cerebellar neurons growing on each substrate exhibited differing growth cone morphologies and rates of neurite extension. Growth of neurites in the presence of cytochalasin B (CB) was not inhibited on substrates of L1 or P84 but was markedly inhibited on N-CAM. Neurons on laminin were initially unable to extend neurites in the presence of CB but recovered this ability within 9 h. These studies suggest that neurite outgrowth mediated by different cell adhesion molecules proceeds via involvement of distinct cytoskeletal interactions. © 1993 John Wiley & Sons, Inc.     

Automated screening of neurite outgrowth

Outgrowth of neurites in culture is used for assessing neurotrophic activity. Neurite measurements have been performed very slowly using manual methods or more efficiently with interactive image analysis systems. In contrast, medium-throughput and noninteractive image analysis of neurite screens has not been well described. The authors report the performance of an automated image acquisition and analysis system (IN Cell Analyzer 1000) in the neurite assay. Neuro-2a (N2a) cells were plated in 96-well plates and were exposed to 6 conditions of retinoic acid. Immunofluorescence labeling of the cytoskeleton was used to detect neurites and cell bodies. Acquisition of the images was automatic. The image set was then analyzed by both manual tracing and automated algorithms. On 5 relevant parameters (number of neurites, neurite length, total cell area, number of cells, neurite length per cell), the authors did not observe a difference between the automated analysis and the manual analysis done by tracing. These data suggest that the automated system addresses the same biology as human scorers and with the same measurement precision for treatment effects. However, throughput of the automated system is orders of magnitude higher than with manual methods.     

Extracellular matrix and neurite outgrowth.

The complex relationship between neuronal cells and the extracellular matrix molecules with which they interact both positively and negatively is currently being investigated on many fronts. Major areas of experimental emphasis include the characterization of an increasing number of extracellular matrix and cell surface associated molecules, the identification of receptors for these molecules, and the analysis of the function of extracellular matrix molecules with respect to neuronal process outgrowth.     

神经细胞粘附分子通过P21活化激酶1促进神经突生长

目的探索神经细胞粘附分子(NCAM)促进神经突生长的分子机制。方法对新生小鼠脑组织行免疫共沉淀以筛选NCAM的结合伴侣。向体外培养的海马神经元中加入免疫共沉淀的阳性筛选分子的抑制剂,观察其对NCAM促进神经突生长作用的影响。提取新生小鼠脑内生长锥以及脂筏,检测NCAM、NCAM的结合伴侣及其上、下游分子在小鼠脑内的空间分布。结果免疫共沉淀发现P21活化激酶1(Pak1)为NCAM的结合伴侣,Pak1抑制剂可以阻断NCAM促进神经突生长的作用。对小鼠脑内脂筏的研究发现NCAM和Pak1上游激活物Pak相互作用交换因子(PIX)、细胞分裂周期蛋白42(Cdc42)在生长锥脂筏上富集,提示NCAM与Pak1的结合以及Pak1的活化可能在脂筏上完成。结论 NCAM通过Pak1途径促进神经突生长,且这一作用的实现可能依赖于脂筏。     

atRA Regulation of NEDD9, a gene involved in neurite outgrowth and cell adhesion

We previously identified NEDD9 (RAINB2/HEF1/Cas-L) as a new downstream target of all-trans retinoic acid (atRA) and its receptors in the human neuroblastoma cell line, SH-SY5Y [R.A. Merrill, A.W.-M. See, M.L. Wertheim, M. Clagett-Dame, Dev. Dyn. 231 (2004) 564-575; R.A. Merrill, J.M. Ahrens, M.E. Kaiser, K.S. Federhart, V.Y. Poon, M. Clagett-Dame, Biol. Chem. 385 (2004) 605-614]. We now provide functional evidence that NEDD9 is directly regulated by atRA through a complex retinoic acid response element (RARE) located in the NEDD9 proximal promoter and consisting of four conserved half-sites separated by 1, 5, and 1 intervening base pairs. We show that a region of the human NEDD9 promoter from −1670 to +15 is sufficient to confer atRA-responsiveness and that a complex RARE located from −475 to −445 is necessary for this effect. While mutation of any one half-site does not eliminate complex formation in electrophoretic mobility shift assays (EMSA); these same mutations, when tested in transient transfection assays, markedly decrease atRA-responsiveness. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that RAR and RXR are bound to the RARE in cells.     

AlphaII-spectrin participates in the surface expression of cell adhesion molecule L1 and neurite outgrowth

AlphaII-spectrin, a basic component of the spectrin-based scaffold which organizes and stabilizes membrane microdomains in most animal cells, has been recently implicated in cell adherence and actin dynamics. Here we investigated the contribution of αΙΙ-spectrin to neuritogenesis, a highly complex cellular process which requires continuous actin cytoskeleton remodeling and cross-talk between extracellular cues and their cell surface receptors, including cell adhesion molecules. Using RNA interference-mediated gene silencing to down-regulate αΙΙ-spectrin expression in human neuroblastoma SH-SY5Y cells, we observed major changes in neurite morphology and cell shape: (1) reduced mean length and a higher number of neurites per cell; occasional long neurites were thinner and displayed abnormal adhesiveness during cell migration resulting in frequent breaks; similar persisting adhesiveness and breaks were also observed in trailing edges of cell bodies; (2) irregular polygonal cell shape in parallel with loss of cortical F-actin from neuronal cell bodies; (3) reduction in protein levels of αΙ- and βΙ-spectrins, but not βΙΙ-spectrin (4) decreased global expression of adhesion molecule L1 and spectrin-binding adapter ankyrin-B, which links L1 to the plasma membrane. Remarkably, αΙΙ-spectrin depletion affected L1 – but not NCAM – cell surface expression, and L1 clustering at growth cones. This study demonstrates that αΙΙ-spectrin is implicated in normal morphology and adhesive properties of neuron cell bodies and neurites, and in cell surface expression and organization of adhesion molecule L1.     

Calmyrin1 binds to SCG10 protein (stathmin2) to modulate neurite outgrowth

Calmyrin1 (CaMy1) is an EF-hand Ca²⁺-binding protein expressed in several cell types, including brain neurons. Using a yeast two-hybrid screen of a human fetal brain cDNA library, we identified SCG10 protein (stathmin2) as a CaMy1 partner. SCG10 is a microtubule-destabilizing factor involved in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels. In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca²⁺-dependent in vivo and requires the C-terminal domain of CaMy1 (residues 99-192) and the N-terminal domain of SCG10 (residues 1-35). CaMy1 did not interact with stathmin1, a protein that is homologous with SCG10 but lacks the N-terminal domain characteristic of SCG10. CaMy1 interfered with SCG10 inhibitory activity in a microtubule polymerization assay. Moreover, CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. This CaMy1 activity did not occur when an N-terminally truncated SCG10 mutant unable to interact with CaMy1 was expressed. Altogether, these data suggest that CaMy1 via SCG10 couples Ca²⁺ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival

Recently, mutations in the progranulin (PGRN) gene were found to cause familial and apparently sporadic frontotemporal lobe dementia (FTLD). Moreover, missense changes in PGRN were identified in patients with motor neuron degeneration, a condition that is related to FTLD. Most mutations identified in patients with FTLD until now have been null mutations. However, it remains unknown whether PGRN protein levels are reduced in the central nervous system from such patients. The effects of PGRN on neurons also remain to be established. We report that PGRN levels are reduced in the cerebrospinal fluid from FTLD patients carrying a PGRN mutation. We observe that PGRN and GRN E (one of the proteolytic fragments of PGRN) promote neuronal survival and enhance neurite outgrowth in cultured neurons. These results demonstrate that PGRN/GRN is a neurotrophic factor with activities that may be involved in the development of the nervous system and in neurodegeneration.     

Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth.

Monoclonal antibodies have been used to identify a 320 kd keratan sulfate proteoglycan that is primarily expressed in the embryonic chick nervous system. Immunohistochemical localization of the proteoglycan shows that it is expressed by putative midline barrier structures in the developing chick central nervous system. When added to laminin or neural cell adhesion molecule that has been adsorbed onto nitrocellulose-coated dishes, the proteoglycan abolishes cell attachment and neurite outgrowth on these adhesive substrata. This effect can be reversed by keratanase treatment and incubation with a monoclonal antibody that recognizes the keratan sulfate chains of the proteoglycan. These data suggest that this neural keratan sulfate proteoglycan plays an important role in the modulation of neuronal cell adhesion during embryonic brain development.     

PC12 cells utilize the homophilic binding site of L1 for cell-cell adhesion but L1-alphavbeta3 interaction for neurite outgrowth

Treatment of PC12 cells with nerve growth factor induces their differentiation into sympathetic neuron-like cells and the concomitant expression of the neural cell adhesion molecule L1, a member of the Ig superfamily. To investigate the mechanism of L1-stimulated neurite outgrowth in PC12 cells, substrate-immobilized fusion proteins containing different extracellular domains of L1 were assayed for their neuritogenic activity. Surprisingly, domain Ig2 of L1, which was previously found to contain both homophilic binding and neuritogenic activities, failed to promote neurite outgrowth. In contrast, L1-Ig6 stimulated neurite outgrowth from PC12 cells. Despite this, homotypic binding of PC12 cells was significantly inhibited by antibodies against L1-Ig2, indicating that L1-L1 binding contributed to the intercellular adhesiveness of PC12 cells, but L1-stimulated neurite outgrowth depends on heterophilic interactions. Thus, PC12 cells provide a valuable model for the study of these two distinct functions of L1. Mutagenesis of L1-Ig6 highlighted the importance of the Arg-Gly-Asp motif in this domain for neuritogenesis. Inhibition studies using cyclic Arg-Gly-Asp-containing peptide and anti-integrin antibodies suggested the involvement of alphavbeta3 integrin. Furthermore, neurite outgrowth stimulated by L1-Ig6 was inhibited by lavendustin A and the MEK inhibitor PD98059, suggesting a signaling pathway that involves tyrosine kinase activation and the mitogen-activated protein kinase cascade.     

Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis

Toll receptors in Drosophila melanogaster function in morphogenesis and host defense. Mammalian orthologues of Toll, the Toll-like receptors (TLRs), have been studied extensively for their essential functions in controlling innate and adaptive immune responses. We report that TLR8 is dynamically expressed during mouse brain development and localizes to neurons and axons. Agonist stimulation of TLR8 in cultured cortical neurons causes inhibition of neurite outgrowth and induces apoptosis in a dissociable manner. Our evidence indicates that such TLR8-mediated neuronal responses do not involve the canonical TLR-NF-kappaB signaling pathway. These findings reveal novel functions for TLR8 in the mammalian nervous system that are distinct from the classical role of TLRs in immunity.     

Tropomodulins are negative regulators of neurite outgrowth

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

Control of neurite outgrowth by RhoA inactivation

cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di-butyric cAMP (db-cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db-cAMP up- and down-regulates GTP-RhoA levels in PC12 cells in a time-dependent manner. Tat-C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)-RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db-cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db-cAMP increases GTP-Rap1 levels, and dominant negative (DN)-Rap1 and DN-Rap-dependent RhoGAP (ARAP3) block neurite outgrowth induced by db-cAMP. DN-p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c-Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db-cAMP.     

Transfected F3/F11 neuronal cell surface protein mediates intercellular adhesion and promotes neurite outgrowth

The mouse neuronal F3 glycoprotein and its chicken homolog F11 belong to a subclass of proteins of the immunoglobulin superfamily with preferential localization on axons and neurites. We have transfected F3 cDNA into CHO cells. Biochemical analysis establishes that the cDNA we have cloned codes for a 130 kd phosphatidylinositol-anchored polypeptide. F3-expressing transfectants exhibited enhanced self-adhesive properties, aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. When used as a culture substrate for sensory neurons, F3-transfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells. The results support the idea that F3/F11 and other closely similar proteins function as cell adhesion molecules that play a role in axonal growth and guidance.     

Rab35 regulates neurite outgrowth and cell shape

Recent studies have identified Rab35 in the endocytic pathway and as a regulator of cytokinesis; however its molecular mechanisms are currently unknown. Here, we find that Rab35 colocalizes with actin filaments and with Cdc42, Rac1 and RhoA, and that Rab35 can activate Cdc42 both in vivo and in vitro. We find activated Rab35 stimulates neurite outgrowth in PC12 and N1E-115 cells via a Cdc42-dependent pathway and that siRNA knockdown of Rab35 activity abolishes neurite outgrowth in these cell lines. We conclude that one function of Rab35 is to regulate Rho-family GTPases and that this role has consequences for neurite outgrowth.

Structured summary

MINT-7012081: Rac1(uniprotkb:P63000) and Rab 35 (uniprotkb:Q15286) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7012070: actin (uniprotkb:P60709) and Rab 35 (uniprotkb:Q15286) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7012095: cdc42 (uniprotkb:P60953) and Rab 35 (uniprotkb:Q15286) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Tenascin-C inhibits beta1 integrin-dependent cell adhesion and neurite outgrowth on fibronectin by a disialoganglioside-mediated signaling mechanism

We have previously shown that the extracellular matrix molecule tenascin-C inhibits fibronectin-mediated cell adhesion and neurite outgrowth by an interaction with a cellular RGD-independent receptor which interferes with the adhesion and neurite outgrowth promoting activities of the fibronectin receptor(s). Here we demonstrate that the inhibitory effect of tenascin-C on beta1integrin-dependent cell adhesion and neurite outgrowth is mediated by the interaction of the protein with membrane-associated disialogangliosides, which interferes with protein kinase C-related signaling pathways. First, in substratum mixtures with fibronectin, an RGD sequence-containing fragment of the molecule or synthetic peptide, tenascin-C inhibited cell adhesion and spreading by a disialoganglioside-dependent, sialidase-sensitive mechanism leading to an inhibition of protein kinase C. Second, the interaction of intact or trypsinized, i.e., cell surface glycoprotein- free, cells with immobilized tenascin-C was strongly inhibited by gangliosides or antibodies to gangliosides and tenascin-C. Third, preincubation of immobilized tenascin-C with soluble disialogangliosides resulted in a delayed cell detachment as a function of time. Similar to tenascin-C, immobilized antibody to GD2 (3F8) or sphingosine, a protein kinase C inhibitor, strongly inhibited RGD- dependent cell spreading. Finally, the degree of tenascin-C-induced inhibition of cell adhesion was proportional to the degree of disialoganglioside levels of expression by different cells suggesting the relevance of such mechanism in modulating integrin-mediated cell- matrix interactions during pattern formation or tumor progression.      

Casein kinase 1 delta functions at the centrosome to mediate Wnt-3a-dependent neurite outgrowth

Septins are conserved guanosine triphosphate-binding cytoskeletal proteins involved in membrane remodeling. In budding yeast, five mitotic septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1), which are essential for cytokinesis, transition during bud growth from a patch to a collar, which splits into two rings in cytokinesis and is disassembled before the next cell cycle. Cdc3, Cdc10, Cdc11, and Cdc12 form an apolar octameric rod with Cdc11 at each tip, which polymerizes into straight paired filaments. We show that Shs1 substitutes for Cdc11, resulting in octameric rods that do not polymerize into filaments but associate laterally, forming curved bundles that close into rings. In vivo, half of shs1Δ mutant cells exhibit incomplete collars and disrupted neck filaments. Importantly, different phosphomimetic mutations in Shs1 can either prevent ring formation or promote formation of a gauzelike meshwork. These results show that a single alternative terminal subunit is sufficient to confer a distinctive higher-order septin ultrastructure that can be further regulated by phosphorylation.     

Glycosaminoglycan-dependent and -independent inhibition of neurite outgrowth by agrin

Agrin is a proteoglycan that can inhibit neurite outgrowth from multiple neuronal types when present as a substrate. Agrin's neurite inhibitory activity is confined to the N-terminal segment of the protein (agrin N150), which contains heparan sulfate (HS) and chondroitin sulfate (CS) side chains. We have examined the activities of various purified recombinant agrin fragments and their glycosaminoglycan (GAG) side chains in neurite outgrowth inhibition. Inhibitory activity was tested using dissociated chick ciliary ganglion neurons or dorsal root ganglion explants growing on laminin or N-cadherin. Initial experiments demonstrated that agrin N150 lacking GAG chains inhibited neurite outgrowth. Both halves of N150, each containing HS and/or CS side chains, could also inhibit neurite growth. Experiments using agrin fragments in which the GAG acceptor residues were mutated, or using agrin fragments purified from cells deficient in GAG synthesis, demonstrated that inhibition by the N-terminal portion of N150 requires GAGs, but that inhibition from the C-terminal part of N150 does not. Thus, the core protein or other types of glycosylation are important for inhibition from the more C-terminal region. Our results suggest that there are two distinct mechanisms for neurite outgrowth inhibition by agrin, one that is GAG-dependent and one that is GAG-independent.