L- and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) inhibit neurite outgrowth from SH-SY5Y cells.

Gangliosides and extracellular matrix molecules influence neurite outgrowth, but the combinatorial effects of these endogenous agents on outgrowth are unclear. Exogenous gangliosides inhibit neurite outgrowth from SH-SY5Y cells stimulated with platelet-derived growth factor-BB, and different isoforms of the ceramide analog threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) stimulate (L-PDMP) or inhibit (D-PDMP) glycosphingolipid biosynthesis. In this study, we determined whether altering the endogenous ganglioside levels with PDMP in SH-SY5Y cells regulates neurite outgrowth on the outgrowth-supporting extracellular matrix molecule, laminin. In cells stimulated with 20 ng/ml platelet-derived growth factor-BB to promote outgrowth, we used image analysis to evaluate neurite outgrowth from SH-SY5Y cells grown on endogenous matrix or laminin and exposed to L- or D-PDMP. Both L- and D-PDMP decreased neurite initiation (the number of neurites/cell, the percent of neurite-bearing cells), elongation (the length of the longest neurite/cell, the total neurite length/cell), and branching (the number of branch points/neurite) from SH-SY5Y cells on endogenous matrix or laminin in a dose-dependent manner in serum-free or serum-containing medium. The inhibitory effects of each PDMP isoform were reversible. Inhibition of neurite outgrowth by L-PDMP could be mimicked by addition of exogenous gangliosides or C2-ceramide. Our analyses of neurite outgrowth in SH-SY5Y cells, a model of developing or regenerating noradrenergic neurons, demonstrate that increasing or decreasing endogenous ganglioside levels decreases neurite outgrowth. These results may indicate that SH-SY5Y cells undergo tight regulation by gangliosides, possibly through modulation of growth/trophic factor- and/or extracellular matrix-activated signaling cascades.

Fibronectin and laminin elicit differential behaviors from SH-SY5Y growth cones contacting inhibitory chondroitin sulfate proteoglycans.

Neuronal growth cones integrate signals from outgrowth-promoting molecules, e.g., laminin (LN) or fibronectin (FN), and outgrowth-inhibiting molecules, e.g., chondroitin sulfate proteoglycans (CSPGs), to navigate through extracellular matrix (ECM). Sensory neurons on LN typically turn to avoid areas rich in inhibitory CSPGs, whereas neuron-like cells of human origin (SH-SY5Y) preferentially stop/stall. These different behaviors may reflect differences in neuron type, response to outgrowth-promoters, or the mechanisms involved in outgrowth vs. inhibition. We used image analysis to determine the effects of different outgrowth promoters on the response of SH-SY5Y cells to inhibitory CSPGs. LN increased neurite initiation and elongation compared to cells plated either on endogenous matrix or FN. On a patterned substratum consisting of alternating stripes of FN and CSPGs, 59.6 +/- 9.3% of SH-SY5Y growth cones turned upon CSPG contact, whereas only 31.9 +/- 8.2% of growth cones turned at a LN/CSPG border. Growth cones on LN spread more upon contact with CSPG than growth cones on FN, whereas growth cones on LN or FN not contacting CSPGs were morphologically similar. Because it is known that integrins are involved in outgrowth on promoters, we analyzed integrin expression in response to inhibitory CSPGs in a choice assay. CSPGs did not induce increases or redistribution of several integrin subunits in SH-SY5Y cells. Furthermore, an anti-beta1 integrin function-blocking antibody did not alter growth cone behavior at a CSPG border. These results indicate that significant mechanistic differences may exist between outgrowth on homogenous outgrowth promoters and growth cone turning at inhibitory molecules.

Nervous system-derived chondroitin sulfate proteoglycans regulate growth cone morphology and inhibit neurite outgrowth: a light, epifluorescence, and electron microscopy study.

Proteoglycans influence aging and plasticity in the nervous system. Particularly prominent are the chondroitin sulfate proteoglycans (CSPGs), which are generally inhibitory to neurite outgrowth. During development, CSPGs facilitate normal guidance, but following nervous system injury and in diseases of aging (e.g., Alzheimer's disease), they block successful regeneration, and are associated with axon devoid regions and degenerating nerve cells. Whereas previous studies used non-nervous system sources of CSPGs, this study analyzed the morphology and behavior of sensory (dorsal root ganglia) neurons, and a human nerve cell model (SH-SY5Y neuroblastoma cells) as they contacted nervous system-derived CSPGs, using a variety of microscopy techniques. The results of these qualitative analyses show that growth cones of both nerve cell types contact CSPGs via actin-based filopodia, sample the CSPGs repeatedly without collapse, and alter their trajectory to avoid nervous system-derived CSPGs. Turning and branching are correlated with increased filopodial sampling, and are common to both neurons and Schwann cells. We show that CSPG expression by rat CNS astrocytes in culture is correlated with sensory neuron avoidance. Further, we show for the first time the ultrastructure of sensory growth cones at a CSPG-laminin border and reveal details of growth cone and neurite organization at this choice point. This type of detailed analysis of the response of growth cones to nervous system-derived CSPGs may lead to an understanding of CSPG function following injury and in diseases of aging, where CSPGs are likely to contribute to aberrant neurite outgrowth, failed or reduced synaptic connectivity, and/or ineffective plasticity.

Neurite outgrowth inhibition by chondroitin sulfate proteoglycan: stalling/stopping exceeds turning in human neuroblastoma growth cones.

Chondroitin sulfate proteoglycan (CSPG) inhibits outgrowth from embryonic chick and rodent neurons in vivo and in vitro and is upregulated during development and following injury. The role of CSPG in outgrowth from human neurons has been largely untested, but is critical for our understanding of regeneration in humans following nervous system injury. Here we determined the effects of CSPG on platelet-derived growth factor (PDGF)-stimulated neurite outgrowth from SH-SY5Y human neuroblastoma cells, a well-accepted model of neuronal differentiation. Cells were plated on glass coverslips adsorbed with laminin (LN), CSPG, or a patterned substratum consisting of alternating stripes of the two molecules. Similar to other studies using chick or rodent neurons, SH-SY5Y cells extend neurites on LN, displaying a 15.2% increase in the total neurite length/cell as compared to cells plated on glass. Cells plated on CSPG alone exhibited reduced neurite outgrowth compared to cells plated on glass or LN. Interestingly, SH-SY5Y growth cones extending on LN and then encountering a CSPG border display more stopping/stalling (62.3%) than turning (27.9%) behaviors. Soluble CSPG inhibits neurite initiation from SH-SY5Y cells plated on glass, but not on LN. These data demonstrate that several CSPG-elicited responses of human neuron-like cells are similar to those from nonhuman neurons. However, approximately 70% of SH-SY5Y growth cones stop or stall at a CSPG border while over 80% of chick sensory neurons turn at a CSPG border. The experimental difference between these models may well indicate a functional difference between animal and human neuronal regeneration.

Gangliosides inhibit growth factor-stimulated neurite outgrowth in SH-SY5Y human neuroblastoma cells.

Exogenously added gangliosides are known to promote neurite outgrowth in a variety of cell types, including some neuroblastoma cell lines. To study neuritogenesis in SH-SY5Y human neuroblastoma we serum starved the cells for 24 hr and exposed them to gangliosides (GM1, GM3, or GT1b), platelet-derived growth factor (PDGF), insulin, nerve growth factor (NGF), insulin-like growth factor I (IGF-I), or combinations of these for 3 days. We measured four parameters of neurite outgrowth using image analysis. PDGF induced neurite outgrowth in SH-SY5Y and GM1 inhibited this. Both phenomena were dose-dependent with neurites/cell and neurite length being below controls with 100 microM GM1, and percent of neurite-bearing cells being below controls with 25, 50, and 100 microM GM1. Similar but more inhibitory results were obtained with GM3 and GT1b. Insulin and IGF-I induced a neuritogenic response that was less potent than that of PDGF and was also inhibited by gangliosides. NGF had no effect on neurite outgrowth but gangliosides were still inhibitory even in cells not treated with growth factors. From this we conclude that gangliosides inhibit spontaneous and trophic factor-induced neurite outgrowth in SH-SY5Y cells. For GM1 and GT1b, but not GM3, this probably involves inhibition of trophic factor receptor function.

Gangliosides inhibit platelet-derived growth factor-stimulated growth, receptor phosphorylation, and dimerization in neuroblastoma SH-SY5Y cells.

SH-SY5Y is a thrice cloned cell line originally derived from the human neuroblastoma cell line SK-N-SH. It grows well in serum-containing medium and undergoes neuritogenesis in response to several trophic factors. Because it has been reported that this clonal line does not have receptors for platelet-derived growth factor (PDGF), it has been unclear what the major mitogenic factor in serum is for these cells. In competitive binding studies using radiolabeled PDGF-BB, we found that SH-SY5Y cells specifically bind PDGF with a KD = 0.14 +/- 0.06 nM and Bmax = 7.3 +/- 2.3 pM. Functionality of these receptors was demonstrated by an increased [3H]-thymidine incorporation in response to PDGF (stimulation index = 2.5). At concentrations of PDGF-BB between 5 and 100 ng/ml, maximum stimulation occurred with 20 ng/ml. Maximum DNA synthesis occurred after 12-24-h exposure to PDGF. Gangliosides GM3 and GT1b greatly inhibited [3H]thymidine incorporation, which was also inhibited to a lesser extent by GM1. Phosphorylation on tyrosine of a 170-kDa protein in response to PDGF stimulation of intact cells was demonstrated by western blot analysis probing with anti-phosphotyrosine antibody. Immunoprecipitation with anti-PDGF beta-receptor antibody and visualization on a western blot with an anti-phosphotyrosine antibody also revealed a 170-kDa protein. Maximum phosphorylation of the 170-kDa protein occurred after 5-min exposure to 20 ng/ml PDGF. This phosphorylation was inhibited by gangliosides GM1, GM2, GD1a, and GT1b but not by GM3. Receptor dimerization was also inhibited by GM1. These results show that SH-SY5Y cells have specific receptors for PDGF-BB that are functional, and can be modulated by gangliosides.