A new agonist of the erythropoietin receptor, Epobis, induces neurite outgrowth and promotes neuronal survival

Apart from its hematopoietic activity, erythropoietin (EPO) is also known as a tissue-protective cytokine. In the brain, EPO and its receptor are up-regulated in response to insult and exert pro-survival effects. EPO binds to its receptor (EPOR) via high- and low-affinity binding sites (Sites 1 and 2, respectively), inducing conformational changes in the receptor, followed by the activation of downstream signaling cascades. Based on the crystal structure of the EPO:EPOR(2) complex, we designed a peptide, termed Epobis, whose sequence encompassed amino acids from binding Site 1. The present study shows that the Epobis peptide specifically binds to EPOR and induces neurite outgrowth from primary neurons in an EPOR-expression dependent manner. Furthermore, Epobis promoted the survival of hippocampal and cerebellar neuronal cultures after kainate treatment and KCl deprivation, respectively. Thus, we identified a new functional agonist of EPOR with the potential to promote neuroregeneration and neuroprotection.     

Nonyloxytryptamine mimics polysialic acid and modulates neuronal and glial functions in cell culture

Polysialic acid (PSA) is a major regulator of cell–cell interactions in the developing nervous system and in neural plasticity in the adult. As a polyanionic molecule with high water‐binding capacity, PSA increases the intercellular space generating permissive conditions for cell motility. PSA enhances stem cell migration and axon path finding and promotes repair in the lesioned peripheral and central nervous systems, thus contributing to regeneration. As a next step in developing an improved PSA‐based approach to treat nervous system injuries, we searched for small organic compounds that mimic PSA and identified as a PSA mimetic 5‐nonyloxytryptamine oxalate, described as a selective 5‐hydroxytryptamine receptor 1B (5‐HT_1B) agonist. Similar to PSA, 5‐nonyloxytryptamine binds to the PSA‐specific monoclonal antibody 735, enhances neurite outgrowth of cultured primary neurons and process formation of Schwann cells, protects neurons from oxidative stress, reduces migration of astrocytes and enhances myelination in vitro. Furthermore, nonyloxytryptamine treatment enhances expression of the neural cell adhesion molecule (NCAM) and its polysialylated form PSA‐NCAM and reduces expression of the microtubule‐associated protein MAP2 in cultured neuroblastoma cells. These results demonstrate that 5‐nonyloxytryptamine mimics PSA and triggers PSA‐mediated functions, thus contributing to the repertoire of molecules with the potential to improve recovery in acute and chronic injuries of the mammalian peripheral and central nervous systems.

     

An NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth and neuronal survival in primary rat neurons

The Neural Cell Adhesion Molecule (NCAM) plays a crucial role in development of the central nervous system regulating cell migration, differentiation and synaptogenesis. NCAM mediates cell-cell adhesion through homophilic NCAM binding, subsequently resulting in activation of the fibroblast growth factor receptor (FGFR). NCAM-mediated adhesion leads to activation of various intracellular signal transduction pathways, including the Ras-mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K)-Akt pathways. A synthetic peptide derived from the second fibronectin type III module of NCAM, the FGL peptide, binds to and induces phosphorylation of FGFR without prior homophilic NCAM binding. We here present evidence that this peptide is able to mimic NCAM heterophilic binding to the FGFR by inducing neuronal differentiation as reflected by neurite outgrowth through a direct interaction with FGFR in primary cultures of three different neuronal cell types all expressing FGFR subtype 1: dopaminergic, hippocampal and cerebellar granule neurons. Moreover, we show that the FGL peptide promotes neuronal survival upon induction of cell death in the same three cell types. The effects of the FGL peptide are shown to depend on activation of FGFR and the MAPK and PI3K intracellular signalling pathways, all three kinases being necessary for the effects of FGL on neurite outgrowth and neuronal survival.     

Participation of protein kinase C alpha isoform and extracellular signal-regulated kinase in neurite outgrowth of GT1 hypothalamic neurons

In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform-selective inhibitors and transfection-based expression of enhanced green fluorescence protein (EGFP)-fused PKC isoforms. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingolTM(a PKCalpha-selective inhibitor), but not by rottlerinTM (a PKCdelta-selective inhibitor), indicating that PKCalpha may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and beta-tubulin. To define the differential localization of PKC isoforms, EGFP-tagged PKCalpha, PKCgamma, and PKCdelta were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKCalpha-EGFP to growth cones and cell-cell adhesion sites, PKCgamma-EGFP to the nucleus, and PKCdelta-EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKCalpha (PKCalpha-Cat-EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKCalpha enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKCalpha-Cat-EGFP alone increased the neurite-outgrowth and doxycycline treatment further augmented the number of neurite-containing cells. We also examined the involvement of the extracellular signal-regulated kinase (ERK) MAP kinase in TPA-induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKCalpha with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase-1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKCalpha isoform-specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway.     

Ceramide influences neurite outgrowth and neuroblastoma cell apoptosis regulated by novel protein kinase C isoforms

We have previously seen that protein kinase C (PKC) epsilon induces neurite outgrowth and that PKCdelta and PKCtheta elicit apoptosis in neuroblastoma cells. In this study we investigate the effects of cell-permeable C(2)-ceramide on these events in SK-N-BE(2) neuroblastoma cells. C(2)-ceramide abolishes neurite formation induced by overexpression of PKCepsilon and, in cells overexpressing PKCdelta or PKCtheta, ceramide treatment leads to apoptosis. Exposure to C(2)-ceramide also suppressed neurite outgrowth induced by retinoic acid, but ceramide did not abrogate neurite induction by treatment with the ROCK inhibitor Y-27632, demonstrating that C(2)-ceramide is not a general inhibitor of neurite outgrowth. The neurite-suppressing effect occurs independently of cell-death. Furthermore, C(2)-ceramide relocated PKCepsilon and the isolated regulatory domain of PKCepsilon from the cytosol to the perinuclear region. In contrast, neither the localization of PKCdelta nor of PKCtheta was affected by C(2)-ceramide. Taken together, the data indicate that the neurite-inhibiting effect of C(2)-ceramide treatment may be caused by a re-localization of PKCepsilon and thus identify a functional consequence of ceramide effects on PKCepsilon localization.     

Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.     

Agonists of fibroblast growth factor receptor induce neurite outgrowth and survival of cerebellar granule neurons

Fibroblast growth factor receptor (FGFR) signaling is pivotal in the regulation of neurogenesis, neuronal differentiation and survival, and synaptic plasticity both during development and in adulthood. In order to develop low molecular weight agonists of FGFR, seven peptides, termed hexafins, corresponding to the β6‐β7 loop region of the FGF 1, 2, 3, 8, 9, 10, and 17, were synthesized. This region shares a homologous amino acid sequence with the FG‐loop region of the second fibronectin Type III module of the neural cell adhesion molecule (NCAM) that binds to the FGFR. Hexafins were shown by surface plasmon resonance to bind to FGFR1‐IIIc‐Ig2‐3 and FGFR2‐IIIb‐Ig2‐3. The heparin analog sucrose octasulfate inhibited hexafin binding to FGFR1‐IIIc‐Ig2‐3 indicating overlapping binding sites. Hexafin‐binding to FGFR1‐IIIc resulted in receptor phosphorylation, but inhibited FGF1‐induced FGFR1 phosphorylation, indicating that hexafins act as partial agonists. Hexafin2, 3, 8, 10, and 17 (but not 1 or 9) induced neurite outgrowth from cerebellar granule neurons (CGNs), an effect that was abolished by two inhibitors of FGFR, SU5402 and inositol hexaphosphate (IP6) and a diacylglycerol lipase inhibitor, RHC‐80267. The neuritogenic effects of selected hexafins could also be inhibited by FGF1 which by itself did not induce neurite outgrowth. Moreover, hexafin1, 3, 9, 10, and 17 (but not 2 or 8) promoted survival of CGNs induced to undergo apoptosis. Thus, selected hexafins induced neuronal differentiation and survival, making them promising pharmacological tools for the study of functional FGFR regulation in development of the nervous system. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Okadaic acid reversibly inhibits neurite outgrowth in embryonic dorsal root ganglion neurons

The aim of this study was to assess the effects of low concentrations of okadaic acid (OA) on neurite outgrowth and cellular integrity in cultures of dissociated dorsal root ganglion (DRG) neurons. The complete and fully reversible arrest of neurite outgrowth was achieved at 1 nM OA, thus ruling out the involvement of protein phosphatase 1 in the observed inhibitory effect. OA at 0.5 nM did not completely block neurite outgrowth, although it reduced the rate of growth by about one third. Protein phosphorylation and the integrity of microtubules and neurofilaments in neuron-enriched cultures were unaffected by 1 nM OA. The rate of synthesis of the low-molecular-weight neurofilament subunit (NFL) was also unchanged by OA treatment. Antimitotic agents used to eliminate proliferating cells did not alter the rate of neurite elongation. Since 1 nM OA does not suffice to inhibit neuronal protein phosphatase 2A fully, owing to the high concentration of this enzyme in neurons, we propose that the inhibitor is affecting a neuronal compartment that contains low levels of the phosphatase. This putative compartment is likely to be located in neurites, which were shown to contain levels of protein phosphatase 2A that were two- to threefold lower than in neuronal perikarya. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 193–201, 1997.     

Structure-based discovery of low molecular weight compounds that stimulate neurite outgrowth and substitute for nerve growth factor

Olanzapine, an atypical antipsychotic drug, was previously shown to protect neuronal cells against nutrient deprivation and to enhance neurite outgrowth. In an effort to identify small molecules with greater potency, the structure of olanzapine was used as a template to search commercially available chemical inventories for compounds with similar features. These compounds were evaluated for their ability to protect cells against glutamine deprivation and low-serum conditions. Positive compounds, 'hits' from initial screening, were then tested for stimulation of neurite outgrowth, alone and in combination with suboptimum concentrations of nerve growth factor (NGF). Numerous neuroprotective compounds (mw < 550 Da) were identified that significantly stimulated neurite outgrowth in PC12 cells. These included 4', 6'-diamidino-2-phenylindole, a nuclear stain; staurosporine, an antibiotic and kinase inhibitor; and 2-phenylamino-adenosine, an adenosine analog. The small molecules were comparable with NGF, and in fact, replaced NGF in outgrowth assays. Pharmacophore analysis of the hits led to the design and synthesis of an active compound, LSU-D84, which represented an initial lead for drug discovery efforts.     

The atRA-responsive gene neuron navigator 2 functions in neurite outgrowth and axonal elongation

Neuron navigator 2 (Nav2) was first identified as an all-trans retinoic acid (atRA)-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, RAINB1) that extend neurites after exposure to atRA. It is structurally related to the Caenorhabditis elegans unc-53 gene that is required for cell migration and axonal outgrowth. To gain insight into NAV2 function, the full-length human protein was expressed in C. elegans unc-53 mutants under the control of a mechanosensory neuron promoter. Transgene expression of NAV2 rescued the defects in unc-53 mutant mechanosensory neuron elongation, indicating that Nav2 is an ortholog of unc-53. Using a loss-of-function approach, we also show that Nav2 induction is essential for atRA to induce neurite outgrowth in SH-SY5Y cells. The NAV2 protein is located both in the cell body and along the length of the growing neurites of SH-SY5Y cells in a pattern that closely mimics that of neurofilament and microtubule proteins. Transfection of Nav2 deletion constructs in Cos-1 cells reveals a region of the protein (aa 837-1065) that directs localization with the microtubule cytoskeleton. Collectively, this work supports a role for NAV2 in neurite outgrowth and axonal elongation and suggests this protein may act by facilitating interactions between microtubules and other proteins such as neurofilaments that are key players in the formation and stability of growing neurites.     

Identification of NCAM-binding peptides promoting neurite outgrowth via a heterotrimeric G-protein-coupled pathway

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I–F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I–F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G-proteins. Neurite outgrowth induced by trans- homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2- and ENFIN11-induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM-dependent manner through G-protein-coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM-mediated signaling.     

Reactive oxygen species regulate F-actin dynamics in neuronal growth cones and neurite outgrowth

Reactive oxygen species are well known for their damaging effects due to oxidation of lipids, proteins and DNA that ultimately result in cell death. Accumulating evidence indicates that reactive oxygen species also have important signaling functions in cell proliferation, differentiation, cell motility and apoptosis. Here, we tested the hypothesis whether reactive oxygen species play a physiological role in regulating F-actin structure and dynamics in neuronal growth cones. Lowering cytoplasmic levels of reactive oxygen species with a free radical scavenger, N -tert-butyl-α-phenylnitrone, or by inhibiting specific sources of reactive oxygen species, such as NADPH oxidases or lipoxygenases, reduced the F-actin content in the peripheral domain of growth cones. Fluorescent speckle microscopy revealed that these treatments caused actin assembly inhibition, reduced retrograde actin flow and increased contractility of actin structures in the transition zone referred to as arcs, possibly by activating the Rho pathway. Reduced levels of reactive oxygen species ultimately resulted in disassembly of the actin cytoskeleton. When neurons were cultured overnight in conditions of reduced free radicals, growth cone formation and neurite outgrowth were severely impaired. Therefore, we conclude that physiological levels of reactive oxygen species are critical for maintaining a dynamic F-actin cytoskeleton and controlling neurite outgrowth.     

Leucine-rich repeat C4 protein is involved in nervous tissue development and neurite outgrowth, and induction of glioma cell differentiation

LRRC4, leucine-rich repeat C4 protein, has been identified in human （GenBank accession No. AF196976）, mouse （GenBank accession No. DQ177325）, rat （GenBank accession No. DQ119102） and bovine （GenBank accession No. DQ 164537） with identical domains. In terms of their similarity, the genes encoding LRRC4 in these four mammalian species are orthogs and therefore correspond to the same gene entity. Based on previous research, and using in situ hybridization, we found that LRRC4 had the strongest expression in hippocampal CA1 and CA2, the granule cells of the dentate gyrus region, the mediodoral thalamic nucleus, and cerebella Purkinje cell layers. Using a P19 cell model, we also found that LRRC4 participates in the differentiation of neuron and glia cells. In addition, extracellular proteins containing both an LRR cassette and immunoglobulin domains have been shown to participate in axon guidance. Our data from neurite outgrowth assays indicated that LRRC4 promoted neurite extension of hippocampal neurons, and induced differentiation of glioblastoma U251 cells into astrocyte-like cells, confirmed by morphology observation and glial fibrillary acidic protein expression.     

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.     

聚唾液酸与唾液酸的研究进展

唾液酸是一族神经氨酸(Neuraminic acid)的衍生物。聚唾液酸(Polysialic acid)是唾液酸(Sialic acid)单体以α-2,8或α-2,9键连接的直链同聚物,是一些哺乳动物细胞中糖蛋白的组成部分和少数几种细菌的胞外多糖组分。综述了唾液酸和聚唾液酸的结构、性质、生物学功能、生物合成和生产应用。     

Heat shock protein 27 in neuronal survival and neurite outgrowth

The small heat shock protein 27 (Hsp27) is well documented to promote neuronal survival in neurodegenerative diseases and following nerve injury. It can directly inhibit apoptotic pathways, and as a chaperone it can ameliorate the toxic effects of misfolded proteins. More recently, Hsp27 has been implicated to also play a role in neurite outgrowth. Thus, Hsp27 is situated at the intersection of neuronal survival and differentiation and, as such, it has dual potential as a key therapeutic target for neuroregeneration.     

Repeated amphetamine treatment induces neurite outgrowth and enhanced amphetamine-stimulated dopamine release in rat pheochromocytoma cells (PC12 cells) via a protein kinase C- and mitogen activated protein kinase-dependent mechanism

Repeated intermittent treatment with amphetamine (AMPH) induces both neurite outgrowth and enhanced AMPH-stimulated dopamine (DA) release in PC12 cells. We investigated the role of protein kinases in the induction of these AMPH-mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). PKC inhibitors chelerythrine (100 nm and 300 nm), Ro31-8220 (300 nm) and the MAP kinase kinase inhibitor, PD98059 (30 micro m) inhibited the ability of AMPH to elicit both neurite outgrowth and the enhanced AMPH-stimulated DA release. The direct-acting PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 250 nm) mimicked the ability of AMPH to elicit neurite outgrowth and enhanced DA release. On the contrary, a selective PKA inhibitor, 100 micro m Rp-8-Br-cAMPS, blocked only the development of AMPH-stimulated DA release but not the neurite outgrowth. Treatment of the cells with acute AMPH elicited an increase in the activity of PKC and MAP kinase but not PKA. These results demonstrated that AMPH-induced increases in MAP kinase and PKC are important for induction of both the enhancement in transporter-mediated DA release and neurite outgrowth but PKA was only required for the enhancement in AMPH-stimulated DA release. Therefore the mechanisms by which AMPH induces neurite outgrowth and the enhancement in AMPH-stimulated DA release can be differentiated.