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.     

Identification of active regions for neurite outgrowth activity of neurocrescin

We previously identified and cloned a neurite outgrowth promoting protein, Neurocrescin (NC), from the extract of the chick denervated leg muscles. In this study, we explored the active region of NC for neurite outgrowth. Using the deletion mutants of NC, we tested their neurite outgrowth activity in the cultured telencephalic neurons of E5 chick embryos. We found three regions which independently had significant neurite outgrowth activity comparable with that of the extract of the chick denervated leg muscles. These regions were not homologous to any well-known active sites such as the laminin active region, IKVAV. In parallel, searching the endogenous deletion mutants of NC in the rat brain, we cloned a mutant in which the region including the larger part of one of the three active regions was deleted. The neurite outgrowth activity of the mutant was significantly lower than that of normal NC. These results suggest the physiological significance of these active regions.     

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.     

Permissive role of Cdc2 activity induced from astrocytes in neurite outgrowth

Following spinal cord injury, glial cells are recognized as major environmental factors hampering axon's regenerative responses. However, recent studies suggested that, in certain circumstances, reactive astrocytes may have a permissive role for axonal regeneration and functional recovery. Here, we report that Cdc2 activation in astrocytes is positively linked to axon growth. Cdc2 was strongly, but transiently, induced from reactive astrocytes within and around the injury cavity. Cdc2 levels in primary, non‐neuronal cells prepared from injured spinal cord were up‐regulated by extending the pre‐injury period. Cdc2‐mediated vimentin phosphorylation was strongly induced in astrocytes after long‐term culture (7 days, LTC) as compared with short‐term culture (3 days, STC). Induction levels of phospho‐vimentin in LTC astrocytes were positively associated with increased neurite outgrowth in co‐cultured dorsal root ganglion neurons. β3 integrin mRNA was induced in LTC astrocytes and activation of β3 integrin was regulated by Cdc2 activity. Furthermore, genetic depletion and pharmacological blockade experiments demonstrate that activation of Cdc2 and β3 integrin in LTC astrocytes is required for neurite outgrowth. Our data suggest that the Cdc2 pathway may play an important role in determining phenotypic expression of astrocytes such that astrocytes provide permissive environments for axonal regeneration following spinal cord injury.     

Thy-1 multimerization is correlated with neurite outgrowth.

Thy-1 is abundantly expressed in the vertebrate nervous system. Perturbation studies in vitro suggest that Thy-1 inhibits neurite outgrowth and stabilizes neuronal processes (N. K. Mahanthappa and P. H. Patterson. (1992). Thy-1 involvement in neurite outgrowth: Perturbation by antibodies, phospholipase C, and mutation. Dev. Biol. 150,47-59). We here report that Thy-1 participates in several types of homophilic interactions, each with differential sensitivity to reduction and boiling. The relative abundance of the multimeric forms of Thy-1 vary with the cell's ability to sprout neurites. Gel filtration chromatography of sympathetic neuron and PC12 cell lysates reveals that Thy-1 immunoreactivity appears in 25-, 45-, and 150-kDa forms. In neurons, Thy-1 immunoreactivity is distributed equally in all three forms, whereas in PC12 cells, the majority of Thy-1 immunoreactivity is found in the higher molecular weight forms. When PC12 cells are induced to sprout neurites with NGF, the Thy-1 size distribution becomes identical to that of neurons. The three forms of Thy-1 immunoreactivity are likely to be homomultimers of Thy-1 because immunoaffinity-purified, soluble Thy-1 also forms complexes similar in size to those found in neuronal extracts. To test whether Thy-1 multimerization may occur through interactions like those between immunoglobulin heavy and light chains, synthetic peptides corresponding to candidate sites for such associations in Thy-1 were tested for their effects on multimerization and neurite outgrowth. One peptide increases the amount of monomeric Thy-1 relative to total Thy-1, and promotes outgrowth. These results suggest that multimeric forms of Thy-1 inhibit process outgrowth and neurite sprouting by stabilizing the surface membrane and/or underlying cytoskeleton.     

RBM4 promotes neuronal differentiation and neurite outgrowth by modulating Numb isoform expression

RBM4 participates in cell differentiation by regulating tissue-specific alternative pre-mRNA splicing. RBM4 also has been implicated in neurogenesis in the mouse embryonic brain. Using mouse embryonal carcinoma P19 cells as a neural differentiation model, we observed a temporal correlation between RBM4 expression and a change in splicing isoforms of Numb, a cell-fate determination gene. Knockdown of RBM4 affected the inclusion/exclusion of exons 3 and 9 of Numb in P19 cells. RBM4-deficient embryonic mouse brain also exhibited aberrant splicing of Numb pre-mRNA. Using a splicing reporter minigene assay, we demonstrated that RBM4 promoted exon 3 inclusion and exon 9 exclusion. Moreover, we found that RBM4 depletion reduced the expression of the proneural gene Mash1, and such reduction was reversed by an RBM4-induced Numb isoform containing exon 3 but lacking exon 9. Accordingly, induction of ectopic RBM4 expression in neuronal progenitor cells increased Mash1 expression and promoted cell differentiation. Finally, we found that RBM4 was also essential for neurite outgrowth from cortical neurons in vitro. Neurite outgrowth defects of RBM4-depleted neurons were rescued by RBM4-induced exon 9–lacking Numb isoforms. Therefore our findings indicate that RBM4 modulates exon selection of Numb to generate isoforms that promote neuronal cell differentiation and neurite outgrowth.     

NCAM induces CaMKIIalpha-mediated RPTPalpha phosphorylation to enhance its catalytic activity and neurite outgrowth

Receptor protein tyrosine phosphatase α (RPTPα) phosphatase activity is required for intracellular signaling cascades that are activated in motile cells and growing neurites. Little is known, however, about mechanisms that coordinate RPTPα activity with cell behavior. We show that clustering of neural cell adhesion molecule (NCAM) at the cell surface is coupled to an increase in serine phosphorylation and phosphatase activity of RPTPα. NCAM associates with T- and L-type voltage-dependent Ca²⁺ channels, and NCAM clustering at the cell surface results in Ca²⁺ influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIα (CaMKIIα). Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM–RPTPα–CaMKIIα complex, resulting in serine phosphorylation of RPTPα by CaMKIIα. Overexpression of RPTPα with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPα activity. Thus, we reveal a novel function for a cell adhesion molecule in coordination of cell behavior with intracellular phosphatase activity.     

A laminin-pepsin fragment with cell attachment and neurite outgrowth activity at distinct sites

Laminin is a large basement membrane glycoprotein which influences the behavior and morphology of a variety of cells. We have found that laminin and a pepsin fragment of laminin (P-lam) contain distinct sites for HT-1080 human fibrosarcoma cell attachment and for neurite outgrowth activity of PC12 and NG108-15 cell lines. Reduction and alkylation of laminin and P-lam fragment disulfide bonds, in the absence of denaturing agents, markedly reduced the cell attachment activity without reducing the neurite outgrowth response. The P-lam fragment (approximately 375 kDa) was found to contain part of the cross region of laminin and a portion of the long arm, on the basis of recognition by antisera against laminin synthetic peptides and fusion proteins. Modification of arginine residues by cyclohexanedione also had no effect on neurite outgrowth but reduced HT-1080 cell adhesion. Modification of lysine residues by succinic and citraconic anhydride, however, abolished laminin neurite outgrowth but not cell attachment activity. Neurite outgrowth activity was recovered by reversing the lysine modification. These data support the existence on laminin of separate sites for cell attachment and for neurite outgrowth.     

Role of spastin and protrudin in neurite outgrowth

Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder characterized by retrograde axonal degeneration that primarily affects long spinal neurons. The gene encoding spastin has a well-established association with HSP, and protrudin is a known binding partner of spastin. Here, we demonstrate that the N-terminal domain of protrudin mediates the interaction with spastin, which is responsible for neurite outgrowth. We show that spastin promotes protrudin-dependent neurite outgrowth in PC12 cells. To further confirm these physiological functions in vivo, we microinjected zebrafish embryos with various protrudin/spastin mRNA and morpholinos. The results suggest that the spinal cord motor neuron axon outgrowth of zebrafish is regulated by the interaction between spastin and protrudin. In addition, the putative HSP-associated protrudinG191V mutation was shown to alter the subcellular distribution and impair the yolk sac extension of zebrafish, but without significant defects in neurite outgrowth both in PC12 cells and zebrafish. Taken together, our findings indicate that protrudin interacts with spastin and induces axon formation through its N-terminal domain. Moreover, protrudin and spastin may work together to play an indispensable role in motor axon outgrowth.     

Enhanced expression and activation of CTP:phosphocholine cytidylyltransferase beta2 during neurite outgrowth

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.     

Dual regulation of RA-RhoGAP activity by phosphatidic acid and Rap1 during neurite outgrowth

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

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.     

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.     

Myosin II activity regulates neurite outgrowth and guidance in response to chondroitin sulfate proteoglycans

Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix in the CNS that inhibit axonal regeneration after CNS injury. Signaling pathways in neurons triggered by CSPGs are still largely unknown. In this study, using well-characterized in vitro assays for neurite outgrowth and neurite guidance, we demonstrate a major role for myosin II in the response of neurons to CSPGs. We found that the phosphorylation of myosin II regulatory light chains is increased by CSPGs. Specific inhibition of myosin II activity with blebbistatin allows growing neurites to cross onto CSPG-rich areas and increases the length of neurites of neurons growing on CSPGs. Using specific gene knockdown, we demonstrate selective roles for myosin IIA and IIB in these processes. Time lapse microscopy and immunocytochemistry demonstrated that CSPGs also inhibit cell adhesion and cell spreading. Inhibition of myosin II selectively accelerated neurite initiation without altering cell adhesion and spreading on CSPGs.     

Gelsolin overexpression enhances neurite outgrowth in PC12 cells.

The rational design of therapies for treating nerve injuries requires an understanding of the mechanisms underlying neurite extension. Neurite motility is driven by actin polymerization; however, the mechanisms are not clearly understood. One actin accessory protein, gelsolin, is involved with remodeling the cytoskeleton, although its role in cell motility is unclear. We report a two-fold upregulation of gelsolin upon differentiation with nerve growth factor. Cells that were genetically modified to overexpress gelsolin have longer neurites and a greater neurite motility rate compared to controls. These data suggest that gelsolin plays an important role in neurite outgrowth.     

Purification of the neurite outgrowth promoting fragment of mouse laminin.

A method for isolation of the neurite outgrowth promoting fragment of mouse laminin (fragment 8) is described in this paper. Besides producing excellent yields, this method was shown to be fast and practical, since it is based on a single step which consists in an ion exchange chromatography of elastase digested laminin.     

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)     

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.     

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.