This study has investigated the possibility that acetylcholinesterase could play a non-classical role as an adhesion factor or growth factor in the development of dopaminergic neurons in organotypic slice culture of postnatal day 1 rats. When the culture medium was supplemented with acetylcholinesterase (3 U/ml), outgrowth of tyrosine hydroxylase-immunoreactive neurites was significantly enhanced. Addition of a specific inhibitor of acetylcholinesterase, BW284c51, caused a decrease in the number of tyrosine hydroxylase neurons and a reduction in the cell body size and extent of neurite outgrowth of remaining neurons. However, echothiophate which also inhibits AChE activity, did not produce these effects. Therefore acetylcholinesterase could act as a growth enhancing factor for dopaminergic neurons, and disruption of an as yet unidentified site on the acetylcholinesterase molecule by BW284c51 could decrease the survival and outgrowth of these neurons.

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Acetylcholinesterase (AChE) is secreted from various brain regions such as the substantia nigra, where levels of this molecule are disproportionately higher than those of choline acetyltransferase. It is thus possible that AChE may have alternative, non-cholinergic functions, one of which could be in development. Indeed, several recent studies have already demonstrated a neurotrophic action of AChE independent of hydrolysis of acetylcholine. In the developing nervous system the dominant forms of AChE differ from the tetramers (G4) that prevail in maturity, in that they are lower molecular weight monomers (G1) and dimers (G2). Therefore, the aims of this study were to explore the neurotrophic role of AChE by comparing the effects of mouse recombinant G1 and G4 AChE on the survival and development of mid-brain tyrosine hydroxylase immunoreactive neurons. Butyrylcholinesterase (BuChE), which also hydrolyses acetylcholine, and basic fibroblast growth factor (bFGF), an established trophic factor for midbrain neurons, were also tested. bFGF had no significant stimulatory effect: moreover, BuChE was also inefficacious, suggesting that the action of AChE was independent of its catalytic site. In contrast, mouse recombinant G1 and G4 AChE both increased the survival as well as the outgrowth of the cultured neurons. However, G1 AChE was more potent than G4 AChE suggesting that developmental forms of AChE exist. The implications of this finding for physiological and pathological functioning of the nervous system are discussed.

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1. In diverse tissues, acetylcholinesterase appears to play a critical role in the functional state of cells completely dependent of cholinergic transmission. However, very little is known about the mechanisms and actual molecular structures mediating the fundamental interactions between this protein and the cellular membrane. 2. In this study, peritoneal macrophages were used as a model system to study the possible interaction between acetylcholinesterase, acting in a non-cholinergic capacity, and the cellular membrane. 3. When acetylcholinesterase was incubated with macrophages harvested from rat peritoneum, the rate of oxygen consumption was increased in a concentration-dependent manner that was independent of mitochondrial block with sodium cyanide. Furthermore, heat inactivation of enzymatic activity or application of BW 284C51 at a concentration which totally blocks catalytic activity did not eliminate the effect. 4. In contrast, incubation with bovine serum albumin or butyrylcholinesterase actually retarded oxygen consumption. 5. The effect of acetylcholinesterase depended on the presence of divalent cations and was inhibited by mannan and D-mannose, but not D-galactose. It is concluded that acetylcholinesterase can induce a "respiratory burst" in macrophages independent of its conventional catalytic site but involving either the mannose receptor of the monocyte-derived macrophage or a possible sugar binding site on acetylcholinesterase itself.