Mouse Acetylcholinesterase Enhances Neurite Outgrowth of Rat R28 Cells Through Interaction With Laminin-1

Abstract

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert ‘non-classical’, morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1.

Regeneration of Aplysia Bag Cell Neurons is Synergistically Enhanced by Substrate-Bound Hemolymph Proteins and Laminin

“…This possibility intrigued us, since AchE is known to bind to laminin4344 and can enhance cell adhesion and neurite outgrowth454647. A synergistic effect of laminin and AchE on neurite outgrowth has also been demonstrated in the R28 neuronal cell line48. These functions are attributed to the peripheral anionic site are unrelated to AchE enzymatic activity4649.…”

Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity

“…Together, these data suggest that ventromedial neurons, such as those that co-express Djchat and the Djche genes, may be important for formation, patterning, and maintenance of the planarian brain. Studies in several animal models and cell culture systems have suggested that AChE may have morphogenic functions during neurodevelopment, which may or may not depend on catalysis of ACh (Bigbee et al 2000; Biagioni et al 2000; Paraoanu et al 2006; Yang et al 2008; Sperling et al 2012; Layer et al 2013). Consistent with the possibility that DjChE activity may regulate planarian brain formation, we previously found that subchronic exposure to high concentrations of the OP chlorpyrifos led to decreased brain size in regenerating but not full worms (Hagstrom et al 2015).…”

An immortalized microglial cell line (Mocha) derived from rat cochlea

“…To place this in context, R28, a sister cell line to Mocha, is a rat retinal precursor cell line established over 20 years ago also using the same 12S E1A immortalization technique (Seigel, 1996). R28 cells, like Mocha cells, do not express all markers of the mature cell type (Seigel et al, 2004), but can respond to some conditions that promote differentiation (Sperling, et al, 2012). R28 cells have been utilized in hundreds of studies, both in vitro and in vivo as a model for cytotoxicity (Patil, et al, 2009), differentiation (Sperling et al, 2012), transplantation (Seigel, et al, 1998) and retinal cell behavior (Uddin, et al, 2016).…”

“…R28 cells, like Mocha cells, do not express all markers of the mature cell type (Seigel et al, 2004), but can respond to some conditions that promote differentiation (Sperling, et al, 2012). R28 cells have been utilized in hundreds of studies, both in vitro and in vivo as a model for cytotoxicity (Patil, et al, 2009), differentiation (Sperling et al, 2012), transplantation (Seigel, et al, 1998) and retinal cell behavior (Uddin, et al, 2016). We anticipate that Mocha cells will show similar utility in studies relating to the inner ear as an immortalized cell line.…”