Protein biomarkers associated with growth and synaptogenesis in a cell culture model of neuronal development

Cerebellar granule cells (CGC) provide a homogenous population of cells which can be used as an in vitro model for studying the cellular processes involved in the normal development of the CNS. They may also be useful for hazard identification as in vitro screens for developmental neurotoxicity. The present study examined morphologic and biochemical markers of CGC neurite outgrowth and synaptogenesis in vitro using both qualitative and quantitative approaches. CGC exhibit a rapid outgrowth of neurites over 14 days in vitro, concomitant with the expression of the synaptic protein Synapsin 1 that was observed as puncta associated with cell bodies and neurites. The expression of neurotypic proteins associated with the cytoskeleton (NF68, MAP2), growth cones (GAP-43) and the synapse (Synapsin I) present an ontogeny that reflects the morphological growth of CGC. The utility of these neurotypic proteins as biomarkers was examined by inhibiting CGC growth using pharmacologic inhibitors of PKC activity and the MAP kinase pathway. Quantitative analysis of neurite outgrowth was performed using an automated image acquisition and analysis system. Treatment of CGC with the MAP kinase pathway inhibitor U0126 significantly decreased total neurite outgrowth, while the inhibitor of classic PKC isoforms Bis I had no effect on this measure. The ontogenetic expression of neurotypic proteins was reduced after treatment with both inhibitors. In particular, Synapsin 1 and GAP-43 expression were both significantly reduced by chemical treatment. These data demonstrate that neurotypic proteins can be used as biomarkers of neuronal development in vitro, and in some cases, may detect changes that are not apparent using morphologic measures.