The long range goal of this project is to understand at the molecular level the mechanisms involved in neural regeneration following trauma. A critical aspect of functional recovery from severe spinal cord injury is axonal sprouting (neuritogenesis). Experimental evidence indicates that several trophic factors, one of which is ganglioside, can stimulate neuritogenesis both in vivo and in vitro. This has generated considerable interest in the possible use of gangliosides as therapeutic agents for clinical treatment of spinal cord injuries. Although the molecular mechanisms through which gangliosides promote neuritogenesis are not known, results of ours and others have shown that gangliosides can affect protein phosphorylation in whole cell and broken cell preparations. Experiments described in this proposal were designed to test our general hypothesis that gangliosides exert their effects on neuritogenesis and neuronal proliferation by modulating the phosphorylation states of specific proteins. The main model that we will use is the SH-SY5Y neuroblastoma cell lines, but confirmatory results of some experiments will be sought using PC12 cells. First, we will study the interactive effects of gangliosides with platelet-derived growth factor (PDGF), insulin-like growth factor-I (IGF-I), and insulin on SH- SY5Y cell proliferation. Second, we will examine the effects of different gangliosides on NGF-stimulated neuritogenesis of SH-SY5Y cells in the presence of PDGF, IGF-I, and insulin. third, we will determine the differential effects of specific gangliosides on three different kinase systems which we propose are critically involved in these biological processes: a) Tyrosine kinase stimulated by PDGF, IGF-I and insulin; b) Protein kinase-C; c) cAMP-dependent protein kinase. Results generated by these experiments should yield considerable insight into the molecular mechanisms through which gangliosides promote neuritogenesis. This knowledge will be of considerable value in understanding neural regeneration studied at the molecular level and in designing new therapeutic strategies for treatment of spinal cord injured patients.
