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ONTOGENY OF PROTEINS ASSOCIATED WITH NEURITE GROWTH AND SYNAPTOGENESIS IN CEREBELLAR GRANULE CELLS IN VITRO.
Citation:
MUNDY, W. R., T. M. FREUDENRICH, B. ROBINETTE, AND C. ROTHERMUND. ONTOGENY OF PROTEINS ASSOCIATED WITH NEURITE GROWTH AND SYNAPTOGENESIS IN CEREBELLAR GRANULE CELLS IN VITRO. Presented at Society of Toxicology, San Diego, CA, March 05 - 09, 2006.
Description:
In vitro techniques may be useful in screening for effects of developmental neurotoxicants. Previously, we characterized changes in biochemical markers associated with neuronal development in a PC12 cell model of differentiation and growth. The current research extended these studies by examining the ontogeny of similar proteins associated with neurite growth and synaptogenesis in a primary neuronal culture. Cerebellar granule cells were prepared from 7-day-old rats and grown on poly-lysine coated 30 mm dishes at high density (for protein extraction) or 96-well plates at low density (for determination of neurite growth). Cells were harvested and lysates prepared on day in vitro (DIV) 0, 1, 2, 4, 5, 7, 8, 10, and 14. Protein expression was determined by western blot or slot. The ontogeny of protein expression varied for the different markers. GAP-43 expression, which is associated with axon elongation, was low on DIV 0, peaked at DIV 4 and then decreased. Expression of the dendritic protein MAP2 increased to DIV 6 and remained high, while the cytoskeletal protein NF68 increased progressively to DIV14. Expression of the synaptic protein Synapsin was virtually absent until DIV 4, then increased to maximal levels at DIV 8 and remained high. To determine whether these proteins were affected by chemical perturbation, we exposed cells at the time of plating to the PKC inhibitor Bis1 at concentrations (2.5 and 5 uM) previously shown to inhibit neurite growth in PC12 cells. Cerebellar granule cells exhibited a wide range of neurite lengths 24 hr after plating (average 57 um) which were decreased by 5 uM Bis1. Effects of Bis1 on proteins were examined on DIV 2, 5, and 8. Bis1 exposure decreased the expression of GAP-43 on DIV 2 and NF68 and Synapsin on DIV 5, but had no effect on MAP2. These data demonstrate the differential ontogeny of several biomarkers corresponding with growth and synaptogenesis in vitro that could be used to study neurotoxicity. (This is an abstract of a proposed presentation and does not necessarily reflect EPA policy).