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EVALUATION OF PROTEIN MARKERS FOR NEURONAL DIFFERENTIATION IN PC12 CELLS.
Citation:
Das, K. P., W R. Mundy, AND T Freudenrich. EVALUATION OF PROTEIN MARKERS FOR NEURONAL DIFFERENTIATION IN PC12 CELLS. Presented at Society of Toxicology, San Francisco, CA, March 25-29, 2001.
Description:
Chemical-induced injury of the developing nervous system can be manifested as a change in the differentiation or growth of neurons. The present study evaluated the use of proteins associated with axonal growth and synaptogenesis as markers for neuronal differentiation in vitro. To establish the expression pattern of proteins associated with growth and synaptogenesis we used an in vitro model, PC12 cells, which upon NGF stimulation differentiate to a sympathetic neuronal phenotype. NGF-induced differentiation (cells with neurite length at least twice the cell body diameter) and neurite growth (length of longest neurite) were determined from photomicrographs using NIH image. Treatment with NGF (50 ng/ml) resulted in differentiation and neurite growth which reached a plateau after 6 days. Expression of GAP-43 (marker of axonal growth) and synapsin (marker of synaptogenesis) were determined in lysates from NGF-treated PC12 cells using PAGE and western blot conditions optimized for each protein. GAP-43 expression was low on day 0 (no NGF) and increased progressively to a maximum on day 5. Synapsin expression increased slowly from day 0-4, then rapidly increased on days 5-7. Because MAP kinase pathways are essential for NGF-induced differentiation, we used the MAP kinase inhibitor U0126 to block the effects of NGF. U0126 (5-30 ?M) decreased differentiation and neurite growth in a concentration-dependent manner. Interestingly, U0126 decreased GAP-43 expression but increased synapsin expression. These results suggest that while GAP-43 expression correlates well with the extent of neurite growth, the regulation of synapsin expression is more complex. (This is an abstract of a proposed presentation and does not necessarily reflect EPA policy).