Identification of neurite outgrowth active sites on the laminin alpha4 chain G domain

The laminin alpha4 chain is widely distributed in various mesodermal tissues, including the perineurium of peripheral nerves, dorsal root ganglion (DRG), skeletal muscle, and capillaries, and plays important roles in synaptic specialization at the neuromuscular junction and in microvascular formation. The C-terminal globular domain (G domain) of the laminin alpha4 chain was previously found to be critical for heparin binding and cell attachment activity. Here, we focused on neurite outgrowth activity of the laminin alpha4 chain G domain. We found that the recombinant alpha4 chain G domain protein (rec-alpha4G) promoted neurite outgrowth of rat pheochromocytoma PC12 cells. When 114 overlapping synthetic peptides that covered the entire G domain were tested for neurite outgrowth activity, nine peptides were active, but the 105 remaining peptides did not exhibit activity. Three of the nine active peptides, A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), and A4G107 (VIRDSNVVQLDV), strongly promoted neurite outgrowth of PC12 cells. A4G107 was found to form amyloid-like fibrils in Congo red, X-ray, and electron microscopy analyses. We also synthesized cyclic peptides to evaluate their conformational requirements. Cyclic peptide A4G82X (cyc-A4G82X;TLFLAHGRLVFX, where X is norleucine) significantly enhanced neurite outgrowth activity, but the rest of the cyclic peptides eliminated the activity. The A4G82 sequence is located on the loop region, suggesting that the activity of A4G82 is required for a loop conformation. These peptides also exhibited neurite outgrowth activity with dorsal root ganglion (DRG) explants and with DRG cells from E14.5 mouse embryos, indicating that they are active in both neuronal cell lines and native neuronal cells. Taken together, the data suggest that the peptides from the laminin alpha4 chain G domain promote neurite outgrowth activity via a specific conformation.     

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.     

Interactions between retinoic acid, nerve growth factor and sonic hedgehog signalling pathways in neurite outgrowth

Many studies have shown a role of retinoid signalling in neurite outgrowth in vitro, and that the retinoic acid receptor (RAR) beta2 is critical for this process. We show here that RARbeta2 is expressed predominantly in dorsal root ganglia (DRG) neuronal subtypes that express neurofilament (NF) 200 and calcitonin gene-related peptide (CGRP), and that these neurons extend neurites in response to RA. We demonstrate that retinoid signalling has a role in neurite outgrowth in vivo, by showing that in a peripheral nerve crush model there is less neurite outgrowth from RARbeta null DRG compared to wild-type. We identify sonic hedgehog (Shh) as a downstream target of the RARbeta2 signalling pathway as it is expressed in the injured DRG of wild-type but not RARbeta null mice. This regulation is direct as when RARbeta2 is overexpressed in adult motoneurons Shh is induced in them. Finally we show that Shh alone cannot induce neurite outgrowth but potentiates RARbeta2 signalling in this process.     

A peptide from the first fibronectin domain of NCAM acts as an inverse agonist and stimulates FGF receptor activation, neurite outgrowth and survival

Neural cell adhesion molecule (NCAM) contributes to axon growth and guidance during development and learning and memory in adulthood. Although the Ig domains mediate homophilic binding, outgrowth activity localizes to two membrane proximal fibronectin-like domains. The first of these contains a site identified as a potential FGF receptor (FGFR) activation motif (FRM) important for NCAM stimulation of neurite outgrowth, but its activity has hitherto remained hypothetical. Here, we have tested the effects of a domain-specific antibody and peptides corresponding to the FRM in cellular assays in vitro. The first fibronectin domain antibody inhibited NCAM-stimulated outgrowth, indicating the importance of the domain for NCAM function. Monomeric FRM peptide behaved as an inverse agonist; low concentrations specifically inhibited neurite outgrowth stimulated by NCAM and cellular responses to FGF2, while saturating concentrations stimulated FGFR-dependent neurite outgrowth equivalent to NCAM itself. Dendrimeric FRM peptide was 125-fold more active and stimulated FGFR activation, FGFR-dependent and FGF-mimetic neurite outgrowth and cell survival (but not proliferation). We conclude that the FRM peptide contains NCAM-mimetic bioactivity accounted for by stimulation of FGF signalling pathways at the level of or upstream from FGF receptors, and discuss the possibility that FRM comprises part of an FGFR activation site on NCAM.     

GDNF promotes neurite outgrowth and upregulates galectin-1 through the RET/PI3K signaling in cultured adult rat dorsal root ganglion neurons

Galectin-1 (GAL-1), a member of a family of β-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG), but its functional role and the regulatory mechanisms of its expression in the peripheral nervous system remain unclear. In the present study, both recombinant nerve growth factor (NGF) and GDNF (50 ng/ml) promoted neurite outgrowth from cultured adult rat DRG neurons, whereas GDNF, but not NGF, significantly increased the number of IB4-binding neurons and the relative protein expression of GAL-1 in the neuron-enriched culture of DRG. The GAL-1 expression in immortalized adult rat Schwann cells IFRS1 and DRG neuron-IFRS1 cocultures was unaltered by treatment with GDNF, which suggests that GDNF/GAL-1 signaling axis is more related to neurite outgrowth, rather than neuron-Schwann cell interactions. The GDNF-induced neurite outgrowth and GAL-1 upregulation were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF may be attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.     

Schwann cells promote neurite outgrowth of dorsal root ganglion neurons through secretion of nerve growth factor

The transplantation of Schwann cells (SCs) could successfully promote axonal regeneration. This is likely to attribute to the adhesion molecules expression and growth factors secretion of SCs. But which factor(s) play a key role has not been precisely studied. In this study, an outgrowth assay using dorsal root ganglia (DRG) neuron-SC co-culture system in vitro was performed. Co-culture of SCs or application of SC-conditioned medium (CM) substantially and significantly increased DRG neurite outgrowth. Further, nerve growth factor and NGF receptor (TrkA) mRNA were highly expressed in Schwann cells and DRG neuron, respectively. The high concentration of NGF protein was detected in SC-CM. When K-252a, a specific inhibitor of NGF receptor was added, DRG neurite outgrowth was significantly decreased in a concentration-dependent manner. These data strongly suggest that SCs play important roles in neurite outgrowth of DRG neurons by secreted NGF.     

rSac3, a novel Sac domain phosphoinositide phosphatase, promotes neurite outgrowth in PC12 cells

Sac domain-containing proteins belong to a newly identified family of phosphoinositide phosphatases （the PIPPase family）. Despite well-characterized enzymatic activity, the biological functions of this mammalian Sac domain PIPPase family remain largely unknown. We identified a novel Sac domain-containing protein, rat Sac3 （rSac3）, which is widely expressed in various tissues and localized to the endoplasmic reticulum, Golgi complex and recycling endosomes, rSac3 displays PIPPase activity with PI（3）P, PI（4）P and PI（3,5）P2 as substrates in vitro, and a mutation in the catalytic core of the Sac domain abolishes its enzymatic activity. The expression of rSac3 is upregulated during nerve growth factor （NGF）-stimulated PC 12 cell neuronal differentiation, and overexpression of this protein promotes neurite outgrowth in PC 12 cells. Conversely, inhibition ofrSac3 expression by antisense oligonucleotides reduces neurite outgrowth of NGF- stimulated PC 12 cells, and the active site mutation of rSac3 eliminates its neurite-outgrowth-promoting activity. These results indicate that rSac3 promotes neurite outgrowth in differentiating neurons through its PIPPase activity, suggesting that Sac domain PIPPase proteins may participate in forward membrane trafficking from the endoplasmic reticulum and Golgi complex to the plasma membrane, and may function as regulators of this crucial process of neuronal cell growth and differentiation.[第一段]     

Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG).

Sulfated proteoglycans (PGs) may play a significant role in the regulation of neurite outgrowth. They are present in axon-free regions of the developing nervous system and repel elongating neurites in a concentration-dependent manner in vitro. The addition of growth-promoting molecules, such as laminin, can modify the inhibitory effect of PGs on neurite outgrowth (Snow, Steindler, and Silver, 1990b). Substrata containing a high-PG/low-laminin ratio completely inhibit neurite outgrowth, while normal, unimpeded outgrowth is observed on low-PG/high-laminin substrata. Therefore, different patterns of neurite outgrowth may result from regulation of the ratio of growth-promoting molecules to growth-inhibiting molecules. Using video microscopy, embryonic chicken dorsal root ganglia neurons (DRG), chicken retinal ganglia neurons (RGC), and rat forebrain neurons (FB) were analyzed as they extended processes from a substratum consisting of laminin alone onto a step gradient of increasing concentrations of chondroitin sulfate proteoglycan (CS-PG) bound to laminin. In contrast to neurite outgrowth inhibition that occurs at the border of a single stripe of high concentration of CS-PG (Snow et al., 1990b and this study), growth cones grew onto and up CS-PG presented in a step-wise graded distribution. Although the behavior of the different cell types was unique, a common behavior of each cell type was a decrease in the rate of neurite outgrowth with increasing CS-PG concentration. These data suggest that appropriate concentrations of growth-promoting molecules combined with growth-inhibiting molecules may regulate the direction and possibly the timing of neurite outgrowth in vivo. The different responses of different neuronal types suggest that the presence of sulfated PG may have varying effects on different aspects of neuronal development.     

Pancreatic islet immunoreactivity to the Reg protein INGAP.

The Reg-related protein family member INGAP (islet neogenesis-associated protein) is a pleiotropic factor enhancing islet neogenesis, neurite growth, beta-cell protection, and beta-cell function. Using an antibody to the N-termini of INGAP, we have identified that immunoreactivity to INGAP localized to the pancreatic endocrine cells in mouse. INGAP- and insulin-immunoreactive cells are mutually exclusive, with INGAP-immunoreactive cells being preserved after streptozotocin-mediated destruction of beta-cells. Glucagon- and INGAP-immunoreactive cells colocalize, although respective antigen expression occurs in different intracellular locations. These data suggest that INGAP-immunoreactive cells include alpha-cells; however, detection of single INGAP-immunoreactive/glucagon-negative cells indicates that this may not be exclusive. In addition to mouse, detection of islet endocrine cells that were INGAP immunoreactive/glucagon immunoreactive/insulin negative was also observed in islets from human, monkey, and rat. These findings reveal that INGAP and/or related group 3 Reg proteins have a conserved expression in the pancreatic islet.     

Anti-Thy-1 antibody-induced neurite outgrowth in cultured dorsal root ganglionic neurons is mediated by the c-Src-MEK signaling pathway

Our previous study has shown that anti-Thy-1 antibody promotes neurite outgrowth of cultured dorsal root ganglion (DRG) neurons in a protein kinase A (PKA)-dependent manner. The present study provided another intracellular signaling pathway for the neurotrophic effect of anti-Thy-1 antibody. In DMSO-treated control cells, Thy-1 was enriched in microdomain-like structures on cell membranes by immunofluorescence observation. Treatment of DRG neurons with anti-Thy-1 antibody not only stimulated neurite outgrowth, but also increased the branching complexity of the neurites in both small and large neurons. We have previously shown that anti-Thy-1 antibody causes a time-dependent activation of mitogen-activated protein kinase (MEK) and of cyclic AMP response-element binding protein (CREB). Here, anti-Thy-1 antibody elicited a transient activation of c-Src kinase, and the activation of c-Src kinase appeared occurring upstream of the activation of MEK and CREB, since pretreatment with the Src kinase inhibitor, PP2, effectively abolished the anti-Thy-1 antibody-induced neurite outgrowth and the phosphorylation of MEK and CREB. CREB phosphorylation might result in upregulation of certain neurite outgrowth-related proteins. We therefore conclude that anti-Thy-1 antibody activates the c-Src kinase-MEK-CREB cascade and overcomes the inhibitory effect of Thy-1 on neurite outgrowth in DRG neurons.     

Neutralization of Schwann Cell-Secreted VEGF Is Protective to In Vitro and In Vivo Experimental Diabetic Neuropathy

The pathogenetic role of vascular endothelial growth factor (VEGF) in long-term retinal and kidney complications of diabetes has been demonstrated. Conversely, little is known in diabetic neuropathy. We examined the modulation of VEGF pathway at mRNA and protein level on dorsal root ganglion (DRG) neurons and Schwann cells (SC) induced by hyperglycaemia. Moreover, we studied the effects of VEGF neutralization on hyperglycemic DRG neurons and streptozotocin-induced diabetic neuropathy. Our findings demonstrated that DRG neurons were not affected by the direct exposition to hyperglycaemia, whereas showed an impairment of neurite outgrowth ability when exposed to the medium of SC cultured in hyperglycaemia. This was mediated by an altered regulation of VEGF and FLT-1 receptors. Hyperglycaemia increased VEGF and FLT-1 mRNA without changing their intracellular protein levels in DRG neurons, decreased intracellular and secreted protein levels without changing mRNA level in SC, while reduced the expression of the soluble receptor sFLT-1 both in DRG neurons and SC. Bevacizumab, a molecule that inhibits VEGF activity preventing the interaction with its receptors, restored neurite outgrowth and normalized FLT-1 mRNA and protein levels in co-cultures. In diabetic rats, it both prevented and restored nerve conduction velocity and nociceptive thresholds. We demonstrated that hyperglycaemia early affected neurite outgrowth through the impairment of SC-derived VEGF/FLT-1 signaling and that the neutralization of SC-secreted VEGF was protective both in vitro and in vivo models of diabetic neuropathy.     

Rapid fibroblast growth factor-induced increases in protein phosphorylation and ornithine decarboxylase activity: regulation by heparin and comparison to nerve growth factor-induced increases.

Fibroblast growth factors (FGFs), like nerve growth factor (NGF), induce morphological differentiation of PC12 cells. This activity of FGF is regulated by glycosaminoglycans. To further understand the mechanisms of FGF and glycosaminoglycan actions in PC12 cells, we studied the regulation of protein phosphorylation and ornithine decarboxylase (ODC) activity by FGF in the presence and absence of heparin. As with NGF, aFGF and bFGF increased the incorporation of radioactive phosphate into the protein tyrosine hydroxylase (TH). The increase in TH phosphorylation was localized to the tryptic peptide, T3. Both T3 and T1 phosphorylations occur in response to NGF, but there was no evidence that aFGF or bFGF stimulated the phosphorylation of the T1 peptide. This result suggests differential regulation of second messenger systems by NGF and FGF in PC12 cells. Heparin, at a concentration that potentiated aFGF-induced neurite outgrowth 100-fold (100 micrograms/ml), did not alter the ability of aFGF to increase S6 phosphorylation or ODC activity. One milligram per milliliter of heparin, a concentration that inhibited bFGF-induced neurite outgrowth, also inhibited bFGF-induced increases in S6 phosphorylation and ODC activity. These observations suggest (i) that acidic and basic FGF activate a protein kinase, possibly protein kinase C, resulting in the phosphorylation of peptide T3 of TH; (ii) that the FGFs and NGF share some but not all second messenger systems; (iii) that heparin potentiates aFGF actions and inhibits bFGF actions in PC12 cells via distinct mechanisms; (iv) that heparin does not potentiate the neurite outgrowth promoting activity of aFGF by enhancing binding to its PC12 cell surface receptor; and (v) that heparin may coordinately regulate several activities of bFGF (induction of protein phosphorylation, ODC and neurite outgrowth) via a common mechanism, most likely by inhibiting the productive binding of bFGF to its PC12 cell surface receptor.     

Neurite outgrowth in PC12 cells. Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (>/=2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub approximately E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub approximately E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of 125 I-labeled beta-lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3). However, Ub-dependent proteolysis of 125I-labeled beta-lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Ub-protein conjugates. Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta-lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Ub-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala. These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.     

Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowth

The IgLONs are a family of glycosyl phosphatidyl inositol-linked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell-cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU-1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU-1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU-1 adhered to a recombinant CEPU-1-Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU-1-Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU-1-Fc, LAMP-Fc and OBCAM-Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON-Fc substrate and overexpression of CEPU-1-GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON-Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non-transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell-cell adhesion and recognition.     

Production and characterization of the recombinant Islet Neogenesis Associated Protein (rINGAP)

Islet Neogenesis Associated Protein (INGAP) is implicated in pancreatic islet neogenesis. INGAP peptide, a pentadecapeptide comprising amino acids 104–118, reverses diabetes in rodents and improves glucose homeostasis in patients with diabetes. The mechanism of INGAP action is unknown, but such studies would benefit from the availability of the full-length recombinant protein (rINGAP).Here we report the production of rINGAP from 293-SF cells following lentiviral transduction, and its characterization by MALDI-TOF and Q-TOF Mass Spectrometry, and HPLC.Importantly, we show that rINGAP exhibits 100× the bioactivity of INGAP peptide on a molar basis in an in vitro assay of human islet regeneration.     

Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM-FGFR-dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM-dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM-responsive axons.