Regenerative failure in the central nervous system (CNS) is a significant clinical problem. Regenerative failure is thought to occur due to the formation of astroglial scar at the site of injury , which in turn blocks regenerating or sprouting neurons. The molecular composition of glial scar, particularly the presence of chondroitin sulfate proteoglycans (CSPGs), is responsible for inhibition of growth cones and regenerative failure. However, the mechanism by which CSPGs inhibit growth cones is not known. Recent successes in various in vivo CNS injury models in eliciting regeneration by digesting the glycosaminoglycan (GAG) portion of CSPGs using chondroitinase ABC suggests that the GAGs present on CSPGs may contribute significantly to CSPG-mediated inhibition. ? ? Our central hypotheses are: i) the CS-GAG component of CSPGs contribute significantly to the inhibition of peripheral (PNS) and CNS growth cones; and that ii) this inhibition is mediated by signaling mechanisms involving an increase in intracellular calcium and a decrease in cAMP levels. The purpose of Aim 1 of this proposal is to characterize the upregulation of CS-GAGs in response to injury of the adult mammalian CNS using a very sensitive and novel technique (FACE). Additionally, Aim l introduces innovative techniques to present the upregulated CS-GAGs to growth cones in a controlled fashion to investigate the direct inhibition of growth cones by CS-GAGs. The purpose of Aim 2 of this proposal is to investigate the signaling mechanism(s) by which CS-GAGs cause growth cone inhibition, focusing specifically on growth cone concentrations of calcium and cAMP and how their levels change when growth cones contact CS-GAGs. ? ? To achieve our goals, we use novel and innovative methods to achieve high spatial control over the immobilization of CSGAGs onto glass cover slips using a micro-fluidics approach. We also use CS-GAGs coupled to Dynal beads to present CSGAGs to growth cones and study the growth cone dynamics in response to contact with inhibitory CS-GAGs. Our methods assume significance because we show that immobilized CS-GAGs are inhibitory whereas soluble GAGs are not, and it is imperative to control the manner in which CS-GAGs are presented to growth cones. This is not surprising given that in vivo, at the site of injury, growth cones encounter CS-GAGs immobilized in the scar matrix. When successfully completed, our research will clarify the extent and mechanism(s) of CS-GAG mediated inhibition of growth cones, and the potential contribution that the GAG components of CSPGs make to CNS inhibition at astroglial scars. With such elucidation, strategies will be developed to alleviate CS-GAG mediated inhibition, assisting growth cones to grow through CS-GAG/CSPG rich areas, facilitating regeneration. ? ?
