Heterologous expression of acetylcholinesterase affects proliferation and glial cytoskeleton of adherent chicken retinal cells

Besides its function at cholinergic synapses, acetylcholinesterase (AChE) e xerts structural functions on neural differentiation, independent of its en zymatic activity. To elucidate such functions, we have previously heterolog ously expressed AChE in histotypic retinal reaggregates, revealing strong e ffects on their histogenesis, particularly on Muller glia processes. To fur ther resolve these findings at a less complex cellular level, in this study we transfected adherent retinal cells of the chick embryo after 2 days i.c . with a sense pSVK3-AChE(rab)-cDNA expression vector encoding for the enti re rabbit AChE gene by calcium phosphate precipitation. Northern blots usin g digoxigenin (DIG)-labeled rabbit cDNA revealed a pronounced level of rabb it AChE mRNA in AChE-transfected cells. Western blot analysis established a n increase in the endogenous AChE protein in transfected cells. Noticeably, AChE activity was not much affected, indicating a post-translational regul ation of overall AChE activity. As a corollary, 5'-bromo-2'-deoxyuridine (B rdU) studies showed a decrease in cell proliferation. Exploring changes of the Muller glia, the cytoskeletal protein vimentin was found to be increase d in transfected cells. Vimentin-stained processes are longer, thicker and more orderly arranged. In conclusion, exogenous expression of rabbit AChE i n chicken retinal mono-layers exerts a structural function on glial cytoske letal organization, independent of AChE activity.