Role of acetylcholinesterase in neurite extension

Work from several laboratories suggests that acetylcholinesterase (AChE) has a role in neuronal differentiation and axonal outgrowth. To evaluate this role, we manipulated AChE enzyme activity and protein expression in two neuronal culture systems: 1) Expiants of dorsal root ganglia (DRG) from E-15 rat fetuses; 2) differentiating murine N1E.115 neuroblastoma cells. AChE activity in DRG was modulated with anticholinesterases and other cholinergic drugs (104M). Both BW 284C51 (selective AChE inhibitor) and eserine (nonselective ChE inhibitor) caused significant decreases in neunte length (25 to 40% at 48 hr), as reported by others. However, the decreases in length after AChE inhibition were not blocked but actually potentiated by cholinergic receptor antagonists (atropine + chlorisondamine). Moreover, there was no correlation between neunte length and AChE activity. Thus AChE activity, per se, was not critical for differentiation. AChE expression was modulated with antisense oligonucleotides in both neuroblastoma and DRG cultures. Antisense oligos directed at the initiation codon or the 3' untranslated region (UTR) caused a sequence-specific and dose-dependent decrease in AChE levels in neuroblastoma cells: 35 uM of 3' UTR antisense reduced cellular AChE activity by 48% at 24 hr and 37% at 48 hr, compared with sense controls. The same dose of 3' UTR sense oligo caused no change, compared with medium-only controls. In DRG, antisense oligonucleotides had smaller but still noticeable effects on AChE activity. Although mean neunte length was not decreased below that in sense-oligo controls, it was lower than in medium only controls, and there was a significant correlation between AChE activity and neunte length. Overall the results are consistent with a supporting role for AChE protein in neurite extension, independent of the classic enzymatic' function.