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Protein Biomarkers Associated With Growth And Synaptogenesis In a cell culture model of neuronal development
Citation:
MUNDY, W. R., B. ROBINETTE, N. RADIO, AND T. M. FREUDENRICH. Protein Biomarkers Associated With Growth And Synaptogenesis In a cell culture model of neuronal development. TOXICOLOGY. Elsevier Science Ltd, New York, NY, 249(2-3):220-229, (2008).
Impact/Purpose:
In vitro models may be useful for the rapid toxicological screening of large numbers of chemicals for their potential to produce toxicity. Such screening could facilitate prioritization of resources needed for in vivo toxicity testing towards those chemicals most likely to result in adverse health effects. Cell cultures derived from nervous system tissue have proven to be powerful tools for elucidating cellular and molecular mechanisms of nervous system development and function, and have been used to understand the mechanism of action of neurotoxic chemicals. Recently, it has been suggested that in vitro models could be used to screen for chemical effects on critical cellular events of neurodevelopment, including neurite growth and synaptogenesis. This manuscript examined morphologic and biochemical markers of CGC maturation in vitro using both qualitative and quantitative approaches. The data demonstrated that neurotypic proteins could be used as biomarkers of neuronal development in vitro, and could detect changes that were not observed using morphologic measures.
Description:
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 I 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 ofCGC. 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 ofCGC with the MAP kinase pathway inhibitor UOl26 significantly decreased total neurite outgrowth, while the inhibitor of classic PKC isomforms Bis I had no effect on this measure. The ontogenetic expression of neurotypic proteins was reduced after treatment with both inhibitors. In particular, Synapsin I expression was greatly reduced by both inhibitors. These data demonstrate that neurotypic proteins can be used as biomarkers of neuronal development in vitro, and in the case of Bis I treatment, detected changes that were not observed using morphologic measures.
