Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons

Abstract

Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChE_T cDNA in either the sense or antisense orientations to overexpress or knock down AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that overexpression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.

Introduction

Establishing the complex cytoarchitecture of the adult nervous system requires a precisely coordinated spatial and temporal expression of many trophic and tropic factors which act to direct several key morphogenic events during this process. While acetylcholinesterase (AChE) is best known as the enzyme that terminates cholinergic transmission by hydrolyzing acetylcholine, an additional morphogenic role for AChE has also been recognized 4, 15, 31, 33, 39. Descriptive anatomical studies have repeatedly documented a transient, peak expression of both AChE mRNA and AChE protein in neurons during periods of axonal outgrowth prior to synaptogenesis, i.e., at a time when the classical cholinolytic function of AChE in synaptic transmission is unnecessary 6, 25, 27, 28, 29, 37, 38, 49. A more specific morphogenic role for AChE, namely, in promoting neurite outgrowth, is supported by studies from our laboratory 10, 11, 41 and others 3, 21, 22, 30, 42, 45, which show that perturbation of surface AChE results in reduced neurite outgrowth. This neuritogenic ability of AChE is independent of its enzymatic activity as first shown by Layer et al. [30] and most recently confirmed by Sternfeld et al. [46], who demonstrated that insertional inactivation of AChE did not affect the neuritogenic capacity of the protein.

This direct neuritogenic ability of AChE has now been convincingly established through genetic manipulation of endogenous AChE expression. Karpel et al. [23] have shown that overexpression of AChE in rat C6 glioma cells induced process extension and cell body enlargement in these otherwise low AChE-expressing cells. Likewise, overexpression of AChE in neuroblastoma cells [24], spinal neurons from Xenopus embryos [46] or retinal neurons [40] resulted in increased process outgrowth that paralleled the increase in AChE expression. Conversely, treatment with AChE antisense cDNA was shown to reduce neurite outgrowth in two neural cell lines 14, 24. Finally, Koenigsberger et al. [24] demonstrated, a direct, positive correlation between the extent of neurite outgrowth and the level of AChE expression by employing a bidirectional modulation of AChE expression in clones of the same neuroblastoma cell line.

Although the mechanism underlying the neuritogenic ability of AChE is not fully understood, this ability of AChE has been shown to occur independently of its catalytic activity 11, 22, 30, 42, 45, 46. Several investigators have hypothesized that the neuritogenic ability of AChE is mediated through an adhesive mechanism based, in part, on the fact that AChE is structurally related to a family of adhesive proteins which include the Drosophila adhesion molecules, neurotactin 7, 19, 44, glutactin [36] and gliotactin [2], and rat neuroligin 20, 43. All of these proteins possess an inactive, extracellular, esterase-like domain which shares amino acid sequence homology and protein structural similarities with AChE. Botti et al. [5] and Felder et al. [12] have described a common annular region of electrostatic negative charge in homologous zones on the surface of these proteins which may participate in protein–protein interactions, and perhaps, adhesion. Based on this structural motif, they have proposed the term “electrotactin” to designate this group of proteins.

The current studies were designed to examine the effects of altered AChE expression on neurite outgrowth in primary dorsal root ganglion (DRG) neurons. To accomplish this, we have employed the adenoviral vector system, a system which has been shown to be an efficient means to achieve high-level, long-term transgene expression in post-mitotic neurons 9, 13, 17, 18, 26, 48. Using adenoviral vectors containing the full-length rat AChE_T cDNA in either sense or antisense orientation, we demonstrate a bidirectional modulation of AChE expression in cultures of primary DRG neurons which directly correlates with the extent of neurite outgrowth from these cells. Since DRG neurons express AChE in vivo while their axons are growing towards their targets prior to synaptogenesis 25, 37, the current findings suggest that AChE on the axonal surface of developing DRG neurons may participate in adhesive interactions during development.