Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including the retina, by different types of stress leading to cellular apoptosis. Here, we tested possible involvement of AChE in
hyperglycemia-induced apoptosis in a
retinal cell line. Y79
retinoblastoma cells were incubated in
starvation media (1% FBS and 1 mg/ml
glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular
glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml. Similar levels of
mannitol were used as control for hyperosmolarity. Cells were harvested at different time intervals for analysis of apoptosis and AChE
protein expression. Apoptosis was detected by the cleavage of
Poly ADP-ribose polymerase (PARP) using western blot, and by
Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay. AChE
protein expression and activity was detected by western blot and by the Karnovsky and Roots method, respectively. Mission(TM)
shRNA for AChE was used to inhibit AChE
protein expression. Treating Y79 cells with 3.5 mg/ml of
glucose, but not with 3.5 mg/ml
mannitol, induced apoptosis which was confirmed by TUNEL assay and by cleavage of PARP. A part of the signaling pathway accompanying the apoptotic process involved up-regulation of the AChE-R variant and an N-extended AChE variant as verified at the
mRNA and
protein level. Inhibition of AChE
protein expression by
shRNA protected Y79 cell from entering the apoptotic pathway. Our data suggest that expression of an N-extended AChE variant, most probably an R
isoform, is involved in the apoptotic pathway caused by
hyperglycemia in Y79 cells.