Effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused growth inhibition of cultured rabbit skin fibroblasts in a dose-dependent manner. At 50 microM both threo and erythro isomers of PDMP completely suppressed the cell growth. Major gangliosides of the fibroblasts, GM3 and GD3, were greatly reduced in amounts in the presence of threo-PDMP and accumulation of ceramides was observed. Surface labeling with galactose oxidase and [3H]NaBH4 demonstrated that neural glycosphingolipids with four or more sugars present on the surface of control cells were not detectable when the fibroblasts were grown in medium containing threo-PDMP. Metabolic labeling of cellular glycosphingolipids with [14C]-galactose showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. In contrast, the erythro isomer of PDMP did not affect the biosynthesis of glycosphingolipids, a result suggesting that the inhibitory effect of erythro-PDMP on cell growth was due to a mechanism other than the inhibition of glucosyltransferase.     

Nonmuscle Myosin Heavy Chain B Is Recognized by a Monoclonal Antibody that Inhibits GM1-Enhanced Neuritogenesis

Abstract: Addition of gangliosides to neuroblastoma cells maintained in vitro has been shown to enhance neuritogenesis. Although the mechanism by which they exert this effect is unknown, it has been postulated that they may act by adhering to cell surface proteins. In this article, we describe the isolation and identification of an S20Y murine neuroblastoma cell protein recognized by a monoclonal antibody that was prepared against putative GM1-binding proteins and shown to inhibit GM1-enhanced neuritogenesis. The protein identified was nonmuscle myosin heavy chain B, which appears to function in neurite formation but may not adhere to gangliosides.     

Neurite outgrowth of neuroblastoma cells: dependence on adhesion surface--cell surface interactions

Neurite outgrowth of C 1300 neuroblastoma cells, which were dispersed from adherent cultures or grown in suspension, was studied on different protein-coated surfaces. Of 29 different surface structures studied, including surfaces treated with various fibronectins, lectins, glycosidases, or glycosyltransferases capable of stimulating fibroblast spreading, only the surfaces coated with plasma fibronectin or with a protein mixture secreted by C6 glioma cells displayed an extensive activity in the sprouting assay. Neurite outgrowth was inhibited by brain gangliosides and by colominic acid (a sialic acid polymer). A 50% inhibition of neurite outgrowth of N18 neuroblasts induced by the glioma cell proteins was observed at the following approximate concentration: 100 microM (0.2 mg/ml) GD1A ganglioside, 20 microM (0.04 mg/ml) GT1B ganglioside, and 5 mg/ml colominic acid. Specificity of inhibition was suggested by the finding that a few polyanionic substances tested were not inhibitory in the sprouting assay, and that the type of gangliosides inhibiting sprouting were found to be major sialoglycolipids of the neuroblasts. A hypothesis is discussed, according to which neurite outgrowth of neuroblasts is stimulated by adhesion involving interactions of the adhesion-mediating protein with cell surface carbohydrates characteristic of brain gangliosides.     

Enhancement of Neurite Outgrowth Following Calpain Inhibition Is Mediated by Protein Kinase C

Abstract: We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell-permeant cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal (“C1”) and calpeptin or by the phospholipid-mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule-associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2-O-tetradecanoylphorbol 13-acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down-regulation following TPA treatment. Cell-free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium-requiring calpain to cleave the SH-SY-5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity.     

Soluble gangliosides in cultured neurotumor cells

Abstract: The biosynthesis and degradation of glycosphingolipids were studied in cytosolic and membrane fractions obtained from rat glioma C6 cells. Both pools had a similar composition of neutral glycosphingolipids but the soluble pool contained only a few percent of the total. The major ganglioside in C6 cells was GM3, of which only 2% was soluble. Whereas the bulk of the membrane GM3 was accessible to surface labeling procedures, the soluble GM3 was not. Mouse neuroblastoma N18 cells also contained small amounts of cytoplasmic gangliosides corresponding to GM3, GM2, GM1, and GDla. When C6 cells were incubated with medium containing [³H]galactose at 37°C, the specific activity of soluble GM3 initially increased more rapidly than that of membrane GM3; by 4 h, the specific activities in both pools became equal. Total incorporation into the membrane pool, however, was always several-fold greater even at the shortest incubation times examined. The labeling pattern of neutral glycosphingolipids in both soluble and membrane fractions indicated the existence of a precursor-product relationship between glucosylceramide and other glycosphingolipids. When labeled cells were transferred to nonradioactive medium, glucosylceramide disappeared the most rapidly, with a 50% loss within <6 h. The turnover rates of other glycosphingolipids were much slower. Although cytosolic GM3 was degraded more rapidly (t_1/2= 26 h) than membrane-bound GM3 (t_1/2= 44 h), its turnover rate was much slower than the time required for transport of GM3 to the cell surface (20–30 min). Our results are consistent with the existence of a small intracellular pool of soluble gangliosides and neutral glycosphingolipids that is stable and independent of the main membrane-bound pool. Although the role of these cytosolic glycolipids is unknown, they do not appear to represent a transport pool between the site of synthesis and the plasma membrane.     

Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition

Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.     

Identification of functional cAMP-dependent protein kinase in a 'neurite minus' mouse neuroblastoma cell line

We have characterized and quantitated the level of cAMP-dependent protein kinase in the NS-20, N1E-115, N-18 and N1A-103 mouse neuroblastoma clonal cell lines, and we have correlated the occurrence of functional cAMP-dependent protein kinase with the dibutyryl cAMP-induced differentiated functions in these cells. Our results demonstrate the presence of functional cAMP-dependent protein kinase in extracts of all four cell lines examined, including the 'neurite minus' N1A-103 cell line. Dibutyryl cAMP induced neurite outgrowth and acetylcholinesterase activity in the NS-20, N1E-115 and N-18 neuroblastoma cell lines, but not in the N1A-103 cell line. However, dibutyryl cAMP caused a 2-3-fold increase in the R1 regulatory subunit protein and cAMP-phosphodiesterase activity in the 'neurite minus' N1A-103 cells in a manner similar to that of the other three 'neurite positive' cell lines. These results suggest that the biochemical lesion(s) subserving the neurite-minus phenotype of the N1A-103 cells may be distal to the activation of cAMP-dependent protein kinase and is in a biochemical pathway distinct from the induction of R1 regulatory subunit protein and cAMP-phosphodiesterase activity.     

Gangliosides Stimulate Calcium Flux in Neuro-2A Cells and Require Exogenous Calcium for Neuritogenesis

The neuritogenic effect of exogenous ganglioside has been documented with a variety of neuronal and neuroblastoma systems, but the mechanism is not understood. Involvement of Ca2+ is suggested by this study which demonstrates that treatment of Neuro-2A cells with bovine brain gangliosides (BBG) in Ca2(+)-depleted medium failed to produce neurite outgrowth. This was in contrast to treatment with retinoic acid or dibutyryl cyclic AMP which induced differentiation under the same conditions. Addition of BBG to Neuro-2A cells caused small, but significant, increases in both influx and efflux of Ca2+. It thus appears that although neuritogenesis can proceed by more than one mechanism, that induced by BBG requires exogenous Ca2+ and involves stimulation of Ca2+ flux.     

Modulation of Neuritogenesis by Ganglioside-Specific Sialidase (Neu 3) in Human Neuroblastoma NB-1 Cells

Plasma membrane-associated sialidase (Neu 3), which specifically hydrolyzes gangliosides, is relatively abundantly present in the nervous system. To understand the role of Neu 3 in neuronal differentiation, we studied the relationship between neurite outgrowth and Neu 3 expression in human neuroblastoma NB-1 cells. The expression of Neu 3 in NB-1 cells increased when neurite outgrowth in these cells was induced by dibutyryl cAMP. While treatment with dibutyryl cAMP alone enhanced the outgrowth of dendrite-like processes, transfection of the Neu 3 gave rise to a more prominent outgrowth of neurites with axon-like characteristics, even in the absence of dibutyryl cAMP. Neu 3 induction by dibutyryl cAMP is probably attributable, in part, to transactivation of the Neu 3 gene through cAMP responsive elements in the 5-upstream region, as revealed by the promotor activity assay using Neu 3 promotor expression plasmid. These results indicate that Neu 3 regulates neurite formation in NB-1 cells, and suggest that this effect may be enhanced by dibutyryl cAMP via a cAMP-dependent pathway.     

Differentiation of neuroblastoma cells induced by an inhibitor of mevalonate synthesis: relation of neurite outgrowth and acetylcholinesterase activity to changes in cell proliferation and blocked isoprenoid synthesis

Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, stimulates neurite outgrowth and acetylcholinesterase (ACE) activity in C1300 (Neuro-2A) murine neuroblastoma cells. Sprouting of neurites began within 4-8 h, before changes in cell proliferation could be detected by [3H]thymidine incorporation or flow cytometry. In contrast, the increase in ACE activity was temporally correlated with suppression of DNA synthesis, which occurred after 8 h. The activity of the membrane marker enzyme phosphodiesterase I was not stimulated by mevinolin. Suppression of protein synthesis with cycloheximide blocked the induction of ACE activity but only partially inhibited neurite outgrowth in the mevinolin-treated cultures. When mevinolin was removed from the culture medium, most of the cells retracted their neurites within 2 h, but ACE activity did not decline until DNA synthesis began to return to control levels after 10 h. Similarly, retraction of neurites in differentiated cells exposed to colchicine was not accompanied by a decrease in ACE activity. DNA histograms suggested that mevinolin arrests neuroblastoma cells in both the G1 and G2/M compartments of the cell cycle. Other cytostatic drugs that arrest cells at different stages of the cell cycle did not cause Neuro-2A cells to form neurites such as those seen in the mevinolin-treated cultures. When incorporation of [3H]acetate into isoprenoid compounds was studied in cultures containing mevinolin in concentrations ranging from 0.25 microM to 25 microM, the labeling of cholesterol, dolichol, and ubiquinone was suppressed by 90% or more at all concentrations. However, significant growth arrest and cell differentiation were observed only at the highest concentrations of mevinolin. Supplementing the medium with 100 microM mevalonate prevented the cellular response to mevinolin, but additions of cholesterol, dolichol, ubiquinone, or isopentenyl adenine were generally ineffective. The cholesterol content of neuroblastoma cells incubated with 25 microM mevinolin for 24 h was not diminished, and protein glycosylation, measured by [3H]mannose incorporation, was decreased only after 24 h at high mevinolin concentration. These studies suggest that the stimulation of neurite outgrowth and the increase in ACE activity induced by mevinolin are independent phenomena. Whereas neurite outgrowth is not related directly to the effects of mevinolin on cell cycling, the induction of ACE is correlated with the inhibition of cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of Ganglioside Biosynthesis in Primary Cultured Neurons

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.     

Gangliosides and neutral glycosphingolipids of normal tissue and oat cell carcinoma of human lung

Concentration and composition of gangliosides and neutral glycosphingolipids of adult human lung, and lung small cell carcinoma were studied. The structures of the glycolipids were determined by quantitative component determination, enzymic degradation, permethylation and fast atom bombardment mass spectrometry. Adult human lung contained mainly gangliosides with lactosylceramide as the basic core, GM3, GD3 and GT3, and approx. equal proportions (10%) of gangliosides of the gangliotetraosyl- and lactotetraosylceramide series. 18 gangliosides with different carbohydrate moieties were identified: four of them were only found in the tumor tissue. The adult human lung contained 85 nmol (77-120) gangliosides and 140 nmol neutral glycosphingolipids per g wet weight. Globoside was the major neutral glycolipid and there were only minor amounts of glycolipids of the lactotetraose series. In small cell carcinoma tissue the concentration of neutral glycosphingolipids was approximately twice as high than in normal lung tissue, and there was a markedly larger concentration of both lactosylceramide and glycolipids of the lactotetraose series and fucose derivatives of these. The concentration of gangliosides varied between 202 and 415 nmol per g wet weight. Compared to normal lung tissue, the tumor tissue had a lower proportion of GD3, and a higher proportion of complex gangliosides, and they contained five tumor-associated gangliosides: Fuc-GM1, Fuc-GD1b, 3'-LM1, Fuc-3'-LM1 and 6'-nLM1.     

Effect of drugs and temperature on biosynthesis and transport of glycosphingolipids in cultured neurotumor cells

Neuroblastoma and glioma cells were grown in the presence of [³H]galactose, and the incorporation of ³H into gangliosides and the transport of newly synthesized gangliosides to the cell surface were examined under different experimental conditions. A variety of drugs, including inhibitors of protein synthesis and energy metabolism, modulators of the cytoskeleton and the ionophore monensin, had no effect on the transport of newly synthesized GD1a in neuroblastoma cells. Only low temperature effectively blocked translocation to the plasma membrane. Monensin, however, had marked effects on the biosynthesis of gangliosides and neutral glycosphingolipids. Whereas incorporation of ³H into complex glycosphingolipids was reduced, labeling of glucosylceramide was increased in cells exposed to monensin. In addition, biosynthesis of the latter glycolipid was less susceptible to low temperatures than that of more complex ones. Previous studies have implicated the Golgi apparatus as the predominant site of glycosylation of gangliosides. As monensin has been reported to interfere with the Golgi apparatus, our results indicate that glucosylceramide may be synthesized at a site that is separate from the site where further glycosylation occurs. Once synthesis of a ganglioside is completed, transport of the molecule to the cell surface proceeds under conditions of cytoskeletal disruption, energy depletion and ionic inbalance, but not low temperature.     

Oligosaccharide Portion of GM1 Enhances Process Formation by S20Y Neuroblastoma Cells

The oligosaccharide portion of ganglioside GM1 was found to enhance neuritogenesis by S20Y murine neuroblastoma cells grown in vitro. The average length of the neurites produced by cells grown in the presence of the oligosaccharide portion of GM1 was comparable to that of cells grown in the presence of intact GM1. The processes of these cells were significantly longer (p less than 0.005, pooled t test) than those of cells grown in the presence of comparable concentrations of sialic acid, lactose, sialyllactose, GD1a, or the oligosaccharide moiety of GD1a. These results suggest that it is the oligosaccharide portion of GM1 that is responsible for the ability of GM1 to enhance process outgrowth by S20Y neuroblastoma cells.     

A PKCbeta isoform mediates phorbol ester-induced activation of Erk1/2 and expression of neuronal differentiation genes in neuroblastoma cells.

Protein kinase C (PKC) activation induces neuronal differentiation of SH-SY5Y neuroblastoma cells. This study examines the role of PKCbeta isoforms in this process. The PKCbeta-specific inhibitor LY379196 had no effect on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced neurite outgrowth from SH-SY5Y neuroblastoma cells. On the other hand, PKCbeta inhibition suppressed the TPA-stimulated increase in neuropeptide Y mRNA, activation of neuropeptide Y gene promoter elements, and phosphorylation of Erk1/2. The TPA-induced increase in neuropeptide Y expression was also inhibited by the MEK inhibitor PD98059. These data indicate that activation of a PKCbeta isoform, through a pathway involving Erk1/2, leads to increased expression of neuronal differentiation genes in neuroblastoma cells.     

Proteoglycans of reactive rat cortical astrocyte cultures: Abundance of N-unsubstituted glucosamine-enriched heparan sulfate

“Reactive” astrocytes and other glial cells in the injured CNS produce an altered extracellular matrix (ECM) that influences neuronal regeneration. We have profiled the glycosaminoglycan (GAG) component of proteoglycans (PGs) produced by reactive neonatal rat cortical astrocytes, and have quantified their neurite-outgrowth inhibitory activity. PGs extracted from cell layers and medium were fractionated on DEAE-Sephacel with a gradient of NaCl from 0.15 to 1.0 M. Monosaccharide analysis of the major peaks eluting at 0.6 M NaCl indicated an excess of GlcNH₂ to GalNH₂, suggesting an approximate HS/CS ratio of 6.2 in the cell layer and 4.2 in the medium. Chondroitinase ABC-generated disaccharide analysis of cell and medium PGs showed a > 5-fold excess of chondroitin 4-sulfate over chondroitin 6-sulfate. Heparin lyase-generated disaccharides characteristic of the highly sulfated S-domain regions within HS were more abundant in cell layer than medium-derived PGs. Cell layer and medium HS disaccharides contained ~ 20% and ~ 40% N-unsubstituted glucosamine respectively, which is normally rare in HS isolated from most tissues. NGF-stimulated neurite outgrowth assays using NS-1 (PC12) neuronal cells on adsorbed substrata of PGs isolated from reactive astrocyte medium showed pronounced inhibition of neurite outgrowth, and aggregation of NS-1 cells. Cell layer PGs from DEAE-Sephacel pooled fractions having high charge density permitted greater NGF-stimulated outgrowth than PGs with lower charge density. Our results indicate the synthesis of both inhibitory and permissive PGs by activated astrocytes that may correlate with sulfation patterns and HS/CS ratios.     

A novel function of transcription factor alpha-Pal/NRF-1: increasing neurite outgrowth

Alpha-Pal/NRF-1 is a critical regulator of the promoter of human IAP/CD47 gene, a gene related to memory formation in rodents. However, its function in neurons was unknown. We found that stable or transient expression of full-length alpha-Pal/NRF-1 in human neuroblastoma IMR-32 cells significantly induced neurite outgrowth and increased the length of neurites both in medium containing 10% fetal bovine serum and in serum-free medium. In contrast, the dominant-negative mutant of alpha-Pal/NRF-1 inhibited the induction and extension of neurites. Ectopic expression of full-length alpha-Pal/NRF-1 also increased the induction of neurite outgrowth in primary mouse cortical neurons. The IAP antisense cDNA significantly inhibited the increase of neurite outgrowth by alpha-Pal/NRF-1. These findings indicate that a novel function of alpha-Pal/NRF-1 is to regulate neuronal differentiation, and that this function is mediated partly via its downstream IAP gene.     

Neurite Extension and Increased Expression of R1 Cyclic AMP-Binding Protein in Ouabain-Resistant Neuroblastoma Mutants

Morphological and biochemical parameters of neuroblastoma differentiation were assessed in 12 clonal derivatives of the N-18 mouse neuroblastoma cell line selected for their ouabain-resistant (ouar) property. When cultured in a normal growth medium, nine of the 12 ouar cell lines exhibited a more complex pattern of neurite outgrowth than the parental N-18 cells. The morphological pattern most frequently observed with the ouar cells was the extension of several branched processes per cell. This pattern of spontaneous neurite outgrowth in the ouar cell lines can be correlated with an increase in expression of the 47,000-dalton RI cyclic AMP (cAMP)-binding protein. The growth rate, intracellular level of cAMP, and acetylcholinesterase activity of the ouar cell lines were not significantly different from those of the parental N-18 neuroblastoma cells. Treatment of the parental and ouar neuroblastoma cell lines with 1 mM N6, O2-dibutyryl cAMP promoted an elaborate pattern of neurite outgrowth and marked increases in acetylcholinesterase and RI cAMP-binding activities. The distinctive pattern of differentiation phenotype exhibited by the ouar cells and the dibutyryl cAMP-induced differentiated neuroblastoma cell suggests that these two protocols yielded different degrees of differentiation. Furthermore, our results suggest a linkage of the biochemical events underlying ouabain resistance and expression of differentiation phenotypes in the mouse neuroblastoma cells.     

Degenerate Human Nucleus Pulposus Cells Promote Neurite Outgrowth in Neural Cells

Innervation of nociceptive nerve fibres into the normally aneural nucleus pulposus (NP) of the intervertebral disc (IVD) occurs during degeneration resulting in discogenic back pain. The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which are associated with stimulation of axonal outgrowth and nociception by neuronal cells, are both expressed by NP cells, with BDNF levels increasing with disease severity. However the mechanism of interaction between human NP cells and neural cells has yet to be fully elucidated. Therefore the aim of this study was to determine whether non-degenerate or degenerate human NP cells inhibit or stimulate neural outgrowth and whether any outgrowth is mediated by NGF or BDNF. Human NP cells from non-degenerate and degenerate IVD were cultured in alginate beads then co-cultured for 48 hours with human SH-SY5Y neuroblastoma cells. Co-culture of non-degenerate NP cells with neural cells resulted in both an inhibition of neurite outgrowth and reduction in percentage of neurite expressing cells. Conversely co-culture with degenerate NP cells resulted in an increase in both neurite length and percentage of neurite expressing cells. Addition of anti-NGF to the co-culture with degenerate cells resulted in a decrease in percentage of neurite expressing cells, while addition of anti-BDNF resulted in a decrease in both neurite length and percentage of neurite expressing cells. Our findings show that while non-degenerate NP cells are capable of inhibiting neurite outgrowth from human neural cells, degenerate NP cells stimulate outgrowth. Neurotrophin blocking studies demonstrated that both NGF and BDNF, secreted by degenerate NP cells, may play a role in this stimulation with BDNF potentially playing the predominant role. These findings suggest that NP cells are capable of regulating nerve ingrowth and that neoinnervation occurring during IVD degeneration may be stimulated by the NP cells themselves.