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doi: 10.1242/10.1242/jcs.00114

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Signalling by glial cell line-derived neurotrophic factor (GDNF) requires heparan sulphate glycosaminoglycan

¹ Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, UK
² Institute of Cell, Animal and Population Biology, King's Buildings, Mayfield Road, Edinburgh, UK

View larger version (49K): [in a new window]   Fig. 1. GAGs are required for responses to GDNF in primary cultures. (a-d) Embryonic dorsal root ganglia form no neurites in the absence of GDNF (a), but form axons quickly in response to 100 ng/ml GDNF (b; arrowheads); inhibition of GAG sulphation with chlorate blocks this response to GDNF (c), but does not block the (stronger) axongenic response to 10 ng/ml NGF (d). (e-h) E10 embryonic intestine develops an extensive network of neurites in culture in response to endogenous GDNF (e); this is reduced by culture in chlorate (f) and inhibited still further by 0.3 U/ml heparinase III (g). Inclusion of 2 mM sulphate in the medium rescues cultures from the effect of chlorate (h) as expected, because chlorate and sulphate compete in the synthesis of the specific donor molecule involved in GAG sulphation.

View larger version (47K): [in a new window]   Fig. 2. GAGs are required for GDNF-induced axonogenesis in PC-12 cells. (a) PC-12 cells cultured in the absence of GDNF remain rounded and form no axons, whereas about 12% of cells form axons when treated with GDNF (b). 30 mM sodium chlorate, a competitive inhibitor of GAG sulphation, blocks this response to GDNF (c), unless sulphate is added to antagonise the effects of chlorate (d). PC-12 cells remain able to produce axons in response to another neurogenic factor, NGF, even in the presence of chlorate (e), showing that GAGs are not required for axon morphogenesis but merely for responsiveness to GDNF. The data from a-d are shown quantitatively in f (NGF-treated PC-12 cells form meshworks of neurites too complicated to count). Total numbers of cells, counted whether or not they bear axons, show no significant variation between these conditions (g).

View larger version (23K): [in a new window]   Fig. 5. Ret autophosphorylation demonstrated by western blotting. (a) Autophosphorylation of c-Ret is barely detectable in the absence of GDNF (-), is strongly induced by 100 ng/ml GDNF (G), but not if cells are pre-treated with 0.1 U/ml heparinase III (G+H). Track H shows control cells treated with just heparitinase III but no GDNF. The bottom panel showing c-Ret regardless of phosphorylation state confirms equal loading of the samples. (b) Confirmation of GFR-1 expression by RET/GFR1-MDCK cells. RET/GFR1-MDCK cells express GFR-1, as expected, in addition to c-Ret (see b), and even normal MDCK cells express a little. The bottom panel, probed for MAP-kinase, is again a loading control.

View larger version (76K): [in a new window]   Fig. 3. Scatter of RET/GFR1-MDCK cells in response to GDNF. Untreated cells (std med) remain as well-bounded islands that are separated by clear spaces (arrows). On addition of GDNF, the inter-island spaces are invaded by motile cells. Incubation of the cells in 30 mM chlorate for the preceding 24 hours and during the scattering period, or incubation in 0.3 U/ml heparanase III for the preceding 2 hours and during the scattering period, greatly reduces this effect of GDNF and clear spaces remain (arrows) although the edges of the islands do still lose their smoothness compared to controls.

View larger version (36K): [in a new window]   Fig. 4. GDNF signalling is blocked by heparinase III but not chondroitinase ABC; (a) RET/GFR1-MDCK cells growing in serum-free medium show little phosphotyrosine at their plasma membranes, but treating them with 100 ng/ml GDNF increases the amount of this phosphotyrosine dramatically (b). Pretreatment of the cells with 0.3 U/ml heparanase III blocks this effect of GDNF (c), although treating them with 0.3 U/ml chondroitinase ABC does not (d). GDNF-induced neuritogenesis by PC12 cells show a similar result (e); cells in plain medium (—) and in medium supplemented with only soluble GFR1 () show only background neuritogenesis, those treated additionally with 100 ng/ml GDNF (G) show significantly enhanced neuritogenesis unless treated throughout the culture period with 0.3 U/ml heparinase III (GH); 0.3 U/ml chondroitinase ABC has no significant effect on neuritogenesis (GC).

View larger version (11K): [in a new window]   Fig. 6. Inhibition of GAG synthesis diminishes binding of 125I-GDNF to the surface of RET/GFR1-MDCK cells. The separation between the curves becomes statistically significant above 300 pM: the plotted points are means of triplicate wells, and error bars represent standard deviation; all point have error bars, but some are too small to see behind the squares and triangles themselves.

View larger version (29K): [in a new window]   Fig. 7. Sensitivity of GDNF signalling to exogenous GAGs. GDNF-induced tyrosine phosphorylation of RET/GFR1-MDCK cells takes place in the presence of 0 (a,d,g), 10 µg/ml (b) and even 100 µg/ml (c) exogenous chondroitin sulphate, although in 100 µg/ml it is somewhat reduced. Heparan sulphate at just 10 µg/ml (e), as well as 100 µg/ml (f) blocks the response. Dermatan sulphate has little effect at 10 µg/ml, although it does, like chondroitin sulphate, reduce the response somewhat at 100 µg/ml. GDNF-induced neuritogenesis in PC-12 cells is also inhibited by exogenous heparan sulphate, even at concentrations as low as 100 ng/ml (j, blue bars); the addition of exogenous heparan sulphate to chlorate-treated cells fails to rescue their response to GDNF at any concentration examined (j, red bars). Error bars represent 95% confidence intervals.

View larger version (45K): [in a new window]   Fig. 8. Heparinase III blocks signalling by 100 ng/ml GDNF (b; compare with the control a). GDNF at the supraphysiological concentration of 1000 ng/ml, however, manages to signal even to cells treated with heparinase III (c)