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

Abstract

Besides its function at cholinergic synapses, acetylcholinesterase (AChE) exerts structural functions on neural differentiation, independent of its enzymatic activity. To elucidate such functions, we have previously heterologously expressed AChE in histotypic retinal reaggregates, revealing strong effects on their histogenesis, particularly on Müller glia processes. To further 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-AChErab-cDNA expression vector encoding for the entire rabbit AChE gene by calcium phosphate precipitation. Northern blots using digoxigenin (DIG)-labeled rabbit cDNA revealed a pronounced level of rabbit AChE mRNA in AChE-transfected cells. Western blot analysis established an increase in the endogenous AChE protein in transfected cells. Noticeably, AChE activity was not much affected, indicating a post-translational regulation of overall AChE activity. As a corollary, 5'-bromo-2'-deoxyuridine (BrdU) studies showed a decrease in cell proliferation. Exploring changes of the Müller glia, the cytoskeletal protein vimentin was found to be increased in transfected cells. Vimentin-stained processes are longer, thicker and more orderly arranged. In conclusion, exogenous expression of rabbit AChE in chicken retinal monolayers exerts a structural function on glial cytoskeletal organization, independent of AChE activity.

Morphoregulation by acetylcholinesterase in fibroblasts and astrocytes

“…5E), T‐1 being nearly three times larger (2437 ± 78 µm 2 ) than parental cells (873 ± 69 µm 2 ), and often extend long AChE‐rich processes over other cells (not shown). Elevation of AChE‐T expression in glioma, neuroblastoma and glial cells also increased cell body size and process extension (Karpel et al, 1996; Bigbee et al, 2000; Keller et al, 2001). A comparison of AChE catalytic activity in parental and AChE‐T‐overexpressing 3T3 clones T‐1 and T‐3 with that of neuronal PC12 cells, in the absence and presence of BW284c51 (Fig.…”

Iron exposure modifies acetylcholinesterase activity in zebrafish (Danio rerio) tissues: distinct susceptibility of tissues to iron overload

“…Our findings showed that the increased activity observed after iron exposure is not a consequence of increased AChE transcript levels. In this sense, it was already demonstrated that AChE from other sources was clearly regulated by a posttranslational event (Robitzki et al 1997;Keller et al 2001).…”

An Overview on the Mechanisms and Applications of Enzyme Inhibition-Based Methods for Determination of Organophosphate and Carbamate Pesticides

“…There were many reports on recombinant AChE. Researchers have cloned AChE cDNA from a variety of organisms, such as fish, Drosophila, chicken, snake, mouse, and nematode, and expressed it exogenously using various expression systems.…”