Growth cone behavior in the presence of soluble chondroitin sulfate proteoglycan (CSPG), compared to behavior on CSPG bound to laminin or fibronectin.
Proteoglycans (PGs) are complex macromolecules of the extracellular matrix (ECM) that have a wide variety of effects on developing and regenerating neurons in vivo and in vitro. One hypothesis regarding the mechanisms of PG regulation of neuronal behavior states that the conformation of PGs may be critical, and thus that ECM- or cell surface-bound PGs may operate differently than secreted (soluble) PGs. Therefore, this study examined differences between the effects of soluble chondroitin sulfate proteoglycan (CSPG) and substratum-bound CSPG on neuronal growth cone behavior. Dissociated chicken dorsal root ganglion (DRG) neurons were cultured on either laminin (LN) or fibronectin (FN), both sensory neurite outgrowth-promotin glycoproteins. CSPG (or chondroitin sulfate alone) was either bound to FN or LN, or was added to the culture media. Subsequently, using time lapse video microscopy and image analysis, this study measured: (1) neuronal attachment, (2) neurite outgrowth, (3) rate of neurite elongation, and (4) filopodial length and lifespan. To determine the site of CSPG action, DRG neurons were grown on either: CS-1, a FN peptide [Humphries M. J. et al. (1987) J. biol. Chem. 262, 6886-6892], or a recombinant FN protein, RFNIIIcs (Maejne, submitted), both of which permit DRG attachment and outgrowth but do not have recognized CSPG binding sites, and the resulting neuronal behavior was compared to that of DRG neurons grown on intact FN. The results of these studies confirm that the effect of CSPG on DRG neurons is concentration-, conformation- and substratum-dependent. On I.N, soluble CSPG had little to no effect on neurite initiation or outgrowth, while substratum-bound CSPG inhibited neurite outgrowth. In contrast, on FN, soluble CSPG inhibited neurite outgrowth and decreased the rate of neurite elongation. Soluble CSPG did not affect the length of sensory growth cone filopodia or filopodial lifespan on either LN or FN. From the FN fragment experiments, we found that: (1) soluble CSPG reduces neurite outgrowth on FN or FN fragments, but not on LN, up to 80%, and reduces elongation rate on FN up to 50%, and (2) soluble CSPG regulates neuronal behavior by binding directly to growth cones elongating on FN. Given that substratum-bound CSPG from a variety of sources is inhibitory to neurite outgrowth and to the rate of neurite elongation, while soluble CSPG often has different effects on growth cone behavior, the regulation of growth cone behavior by CSPGs may be dependent upon CSPG conformation. Further, CSPG may affect growth cone behavior by either binding to the substratum or by binding directly to growth cones.
Snow
D M
DM
Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis 55455, USA.
Brown
E M
EM
Letourneau
P C
PC
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D002809:Chondroitin Sulfates / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism / Q000502:physiology; D005353:Fibronectins / Q000378:metabolism*; D005727:Ganglia, Spinal / Q000166:cytology / Q000254:growth & development; D007797:Laminin / Q000378:metabolism*; D016501:Neurites / Q000502:physiology / Q000648:ultrastructure; D011509:Proteoglycans / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology* / Q000648:ultrastructure
D005353:Fibronectins; D007797:Laminin; D011509:Proteoglycans; D002809:Chondroitin Sulfates