Antisense Inhibition of R-Cognin Expression Modulates Differentiation of Retinal Neurons In Vitro

Purpose: Retina cognin (R-cognin) is a 50 kDa membrane-associated polypeptide expressed during retinogenesis where it is involved in mediating tissue-specific cell-cell interactions. In addition to its intercellular role in aggregation, Rcognin may act as a cell surface signaling molecule. An antisense oligonucleotide was used to inhibit R-cognin expression and to investigate the effects of this inhibition on subsequent neuronal differentiation. Methods: Cultures of retina cells were prepared from 6 day (E6) and 8 day (E8) chicken embryos and were incubated with a deoxyoligonucleotide complEmentary to 20 bases of the sequence encoding R-cognin or random oligonucleotides. The levels of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD), markers of cholinergic and GABAergic differentiation, respectively, were detected by Western blots on protein extracts from treated cultures. Results: The antisense treatment inhibited ChAT levels at E6 and GAD levels at E8. The treatment resulted in no decrease in the level of the enzyme glyceraldehyde 3-phosphate dehydrogenase. A random oligonucleotide did not affect the levels of any of the proteins. Conclusions: These results confirm the cell recognition role of R-cognin and suggest that it is important in intracellular signaling cascades necessary for normal retina development.