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. Author manuscript; available in PMC: 2009 Apr 1.
Published in final edited form as: Toxicol Appl Pharmacol. 2007 Nov 17;228(1):32–41. doi: 10.1016/j.taap.2007.11.005

Chlorpyrifos and Chlorpyrifos-Oxon Inhibit Axonal Growth by Interfering with the Morphogenic Activity of Acetylcholinesterase

Dongren Yang 1,*, Angela Howard 2,*, Donald Bruun 1, Mispa Ajua-Alemanj 3, Cecile Pickart 3,§, Pamela J Lein 1,2,†
PMCID: PMC2408880  NIHMSID: NIHMS44815  PMID: 18076960

Abstract

A primary role of acetylcholinesterase (AChE) is regulation of cholinergic neurotransmission by hydrolysis of synaptic acetylcholine. In the developing nervous system, however, AChE also functions as a morphogenic factor to promote axonal growth. This raises the question of whether organophosphorus pesticides (OPs) that are known to selectively bind to and inactivate the enzymatic function of AChE also interfere with its morphogenic function to perturb axonogenesis. To test this hypothesis, we exposed primary cultures of sensory neurons derived from embryonic rat dorsal root ganglia (DRG) to chlorpyrifos (CPF) or its oxon metabolite (CPFO). Both OPs significantly decreased axonal length at concentrations that had no effect on cell viability, protein synthesis or the enzymatic activity of AChE. Comparative analyses of the effects of CPF and CPFO on axonal growth in DRG neurons cultured from AChE nullizygous (AChE−/−) versus wildtype (AChE+/+) mice indicated that while these OPs inhibited axonal growth in AChE+/+ DRG neurons, they had no effect on axonal growth in AChE−/− DRG neurons. However, transfection of AChE−/− DRG neurons with cDNA encoding full-length AChE restored the wildtype response to the axon inhibitory effects of OPs. These data indicate that inhibition of axonal growth by OPs requires AChE, but the mechanism involves inhibition of the morphogenic rather than enzymatic activity of AChE. These findings suggest a novel mechanism for explaining not only the functional deficits observed in children and animals following developmental exposure to OPs, but also the increased vulnerability of the developing nervous system to OPs.

Keywords: organophosphorus pesticides, organophosphates, chlorpyrifos, axonal growth, acetylcholinesterase, developmental neurotoxicity, in vitro models

INTRODUCTION

There is increasing public and regulatory concern that exposure to low levels of organophosphorus pesticides (OPs) may interfere with neurodevelopment in children (Costa, 2006). This concern was initially triggered by animal studies demonstrating that the developing nervous system is more susceptible than the mature nervous system to the neurotoxic effects of OPs(Pope et al., 1991; Pope and Chakraborti, 1992; Mortensen et al., 1998; Moser et al., 1998; Moser and Padilla, 1998), and by documentation of widespread exposure of children to OPs in both rural and urban environments (Davis and Ahmed, 1998; Eskenazi et al., 1999; Landrigan et al., 1999; Adgate et al., 2001; Lu et al., 2001; Whyatt and Barr, 2001; CDC, 2003; Curl et al., 2003; Barr et al., 2004). Recent epidemiological studies indicating a link between exposure to low levels of OPs and neurobehavioral deficits in infants (Engel et al., 2007) and children (Rohlman et al., 2005; Kofman et al., 2006; Eskenazi et al., 2007; Lizardi et al., 2007) further heighten this concern, and underscore the need to better understand the cellular and molecular mechanism(s) of OP developmental neurotoxicity (Costa, 2006).

In experimental animal models, perinatal exposure to the OP chlorpyrifos (CPF) causes cognitive and behavioral deficits in the absence of significant inhibition of the enzymatic activity of acetylcholinesterase (AChE, EC 3.1.1.7) or downregulation of cholinergic receptors (Jett et al., 2001; Levin et al., 2001; Levin et al., 2002), both mechanisms thought to mediate OP neurotoxicity following high level exposures (Ecobichon, 1994; Abou-Donia, 2003; Costa, 2006). These data have been widely interpreted to mean that OPs target molecules other than AChE to cause developmental neurotoxicity (Slotkin, 2004; Casida and Quistad, 2005; Costa, 2006). However, evidence demonstrating that AChE functions to promote axonal growth in normally developing neurons (Bigbee et al., 1999; Brimijoin and Koenigsberger, 1999; Grisaru et al., 1999) suggest an alternative interpretation in which OP developmental neurotoxicity is mediated by disruption of the morphogenic rather than enzymatic activity of AChE. This hypothesis is supported by demonstrations that various pharmacological cholinesterase inhibitors block AChE-induced axonal growth at concentrations significantly below those that inhibit the enzymatic activity of AChE (Dupree and Bigbee, 1994; Dupree and Bigbee, 1996; Johnson and Moore, 1999; Munoz et al., 1999). Since precise regulation of the rate and direction of axonal growth is essential to the development of functional neural circuits (Berger-Sweeney and Hohmann, 1997; Cremer et al., 1998; Barone et al., 2000), interference with the axon-promoting activity of AChE represents a biologically plausible mechanism for explaining not only the functional deficits observed in children and animals exposed to OPs during development but also the increased vulnerability of the developing nervous system to OPs.

There is experimental evidence that OPs perturb normal patterns of axonal growth in the developing nervous system. Perinatal exposure to OPs alters both brain morphometry (Veronesi and Pope, 1990; Campbell et al., 1997; U.S.E.P.A., 2000; Roy et al., 2004) and the ratio of membrane protein to total protein in the brain, which was used by the authors as a surrogate measure of neurite outgrowth (Qiao et al., 2003; Slotkin et al., 2006). In vitro, CPF has been shown to inhibit neurite outgrowth in neural cell lines (Li and Casida, 1998; Song et al., 1998; Das and Barone, 1999; Sachana et al., 2001; Hargreaves et al., 2006) and axonal growth in primary neuronal cell cultures (Howard et al., 2005). These dysmorphogenic effects were observed in vitro at OP concentrations that did not inhibit AChE enzymatic activity, indicating that OPs alter axonal growth independent of effects on AChE enzymatic activity. However, these studies did not directly address the question of whether the axon inhibitory effects of OPs required AChE, which is a requisite step for determining whether OPs interfere with the morphogenic activity of AChE.

To test this hypothesis, we determined whether genetic deletion of AChE influenced the effects of CPF and its oxon metabolite (CPFO) on axonal growth in primary cultures of sensory neurons derived from the dorsal root ganglia (DRG). DRG neurons were chosen for these studies primarily because the axons and axonal growth cones of DRG express AChE in vivo and in vitro during periods of axonal growth (Cochard and Coltey, 1983; Biagioni et al., 1989; Oudega and Marani, 1990; Koenigsberger et al., 1998), and cultured DRG neurons have been used extensively to characterize the morphogenic activity of AChE (Dupree and Bigbee, 1994; Dupree and Bigbee, 1996; Sharma and Bigbee, 1998; Bigbee et al., 2000; Sharma et al., 2001). Other advantages of using cultured DRG neurons for these studies included: (1) DRG neurons extend only axons both in vivo and in vitro thereby eliminating the need to distinguish between OP effects on axons versus dendrites, which we have previously shown are different (Howard et al., 2005); (2) Cultured DRG neurons are not cholinergic (Schoenen et al., 1989; Chauvet et al. 1995), which removes the confound of potential OP effects on cholinergic receptors or acetylcholine levels; and (3) DRG yield a relatively homogenous population of neurons that can be cultured in the absence of serum (Kleitman et al., 1998), which is a significant source of AChE. In addition, sensory functions mediated by DRG neurons are compromised in AChE null mice (Duysen et al., 2001) and in humans exposed chronically to low levels of OPs (Ray and Richards, 2001). To assess the effects of genetic deletion of AChE on the response to OPs, DRG neurons were cultured from AChE null mice, which express no AChE as a result of targeted deletion of exons 2, 3, 4 and 5 of AChE by homologous recombination (Xie et al.,1999). Our data indicate that in DRG neurons expressing wildtype AChE levels, CPF and CPFO inhibit axonal growth independent of effects on cell viability, protein synthesis or AChE enzymatic activity. In contrast, CPF and CPFO have no effect on axonal growth in DRG neurons derived from AChE null mice, but expression of full-length AChE restores the wildtype response to OPs in AChE null neurons. These data support the hypothesis that these OPs alter morphogenic events critical to establishing neuronal connectivity in part by interfering with the morphogenic activity of AChE.

MATERIALS AND METHODS

Cell culture

All procedures involving animals were performed according to protocols approved by the Johns Hopkins University and Oregon Health & Science University Institutional Animal Care and Use Committees. Following published protocols (Kleitman et al., 1998), sensory neurons were dissociated from the dorsal root ganglia (DRG) of embryonic day 15 (E15) Holtzman rats (Harlan, Indianapolis, IN) or E14 AChE+/+ and AChE−/− mouse pups (AChE +/− breeding pairs were generously provided by Oksana Lockridge, University of Nebraska Medical Center). Mice were genotyped by RT-PCR as previously described (Xie et al., 1999). DRG neurons were plated onto glass coverslips precoated with poly-D-lysine (100µg/ml, Sigma, St. Louis, MO) and maintained in serum-free medium supplemented with β-NGF (100ng/ml, Harlan Bioproducts, Indianapolis, IN) as previously described (Higgins et al., 1991).

COS7 cells were purchased from American Type Culture Collection (Manassas, VA) and maintained as recommended by the vendor. COS7 cells were adapted to grow in serum-free VPSFM Medium (Invitrogen) prior to use in the experiments described in this study.

Transfection of cell cultures

Full-length human AChE cloned into a pGS expression plasmid (pAChE) (Lockridge et al., 1997) was a gift from Dr. Oksana Lockridge (University of Nebraska Medical Center). Plasmid expressing catalytically inactive AChE (pAChEΔ) in which the AGC codon encoding the active site serine residue (Ser203) was mutated to GCC to encode Ala was generated by mutagenic whole plasmid PCR of pAChE. The open reading frame of the mutant AChEΔ construct was sequenced in its entirety to verify that the Ser203→Ala203 mutation was the only mutation present in the plasmid.

Cultured DRG neurons (100 cells/mm2) and COS7 cells (120 cells/mm2) were transfected with GFP plasmid (pGFP, Clontech, Palo Alto, CA) only, or co-transfected with pGFP and either pAChE or pAChEΔ. Adherent COS7 cells were transiently transfected using the Trans IT-LT1 transfection reagent as described by the manufacturer (Mirus, Madison WI). Dissociated DRG neurons were transfected by electroporation prior to plating using the Amaxa Nucleofector kit (Amaxa Biosystems, Germany) according to the manufacturer’s instructions.

Pesticide treatment

Chlorpyrifos (O, O-diethyl O-phosphorothionate, 99.5% pure) and CPF-oxon (CPFO, 98.5% pure) were purchased from Chem Service (West Chester, PA) and stored as recommended by the manufacturer. Stock solutions of CPF and CPFO were prepared in 100% DMSO. Stocks were diluted 1:1,000 in culture medium to give final concentrations of CPF and CPFO as indicated in the text. Cultures used as vehicle controls were treated with culture medium supplemented with DMSO at a 1:1,000 final dilution. Previous studies indicated that this amount of DMSO did not alter axonal outgrowth relative to medium alone (Howard et al., 2005). Experiments were initiated in untransfected cultures 1h after plating and in transfected cultures 24h after electroporation by exchanging the existing medium in the culture for freshly prepared medium containing CPF or CPFO. During this process, less than 5% of the cells were lost due to poor adherence to the culture dish. Cultures were exposed to pesticide for 24h.

Morphometric analyses

At the conclusion of experimental treatments, cultured DRG neurons were fixed with 4% paraformaldehyde and immunostained for the neuronal antigen, protein gene product 9.5 (PGP 9.5; Biogenesis Inc. Brentwood, NH), or for the axon specific antigen, phosphorylated forms of the M and H neurofilaments (Sternberger-Meyer Immunochemicals, Jarrettsville, MD). Antigen-antibody complexes were detected by indirect immunofluorescence as previously described. (Lein et al., 1995) Fluorescent cell images were captured using a Spot digital camera, and an observer blinded to the experimental conditions quantified axonal length using Spot imaging software (Diagnostic Instruments Inc., Sterling Heights, MI). Processes were identified as axons if they were immunopositive for either PGP9.5 or phosphorylated neurofilaments, exhibited a distinct growth cone and their length was ≥ the diameter of the cell body. Axonal growth was evaluated in a minimum of 45 randomly chosen neurons from three different cultures (≥15 neurons per culture) of each experimental condition. Each experimental result was confirmed in cultures obtained from three independent dissections; all data presented in the text are from a single representative culture series.

Cell viability

Following a 24h treatment with pesticides, cell viability was measured using calcein AM and propidium iodide (Molecular Probes, Eugene, OR) as previously described (Howard et al., 2005). The number of viable and nonviable cells was visualized at 100X in 6 randomly chosen fields per experimental condition, and data are expressed as the percent viable cells per field.

Analysis of protein synthesis

Protein synthesis was determined by quantifying 3H-leucine uptake in cultured DRG neurons as previously described (Lein and Higgins, 1991). Briefly, DRG neurons were plated at a density of 400 cells/mm2 on 35mm dishes precoated with ammoniated rat tail collagen using a published protocol (Higgins et al, 1991). Four hours after plating, cultures were exposed to medium supplemented with 3H-leucine (50µCi/ml) in the absence or presence of varying concentrations of CPF. After an overnight incubation at 37°C, cultures were washed with HBSS supplemented with 4mM Ca2+ and 100µM cold leucine then solublized with NaOH (1M). Total uptake of 3H-leucine from 3 replicate dishes per experimental condition was quantified using a liquid scintillation counter (Beckman Coulter, Fullerton CA). Background radioactivity was determined in sister cultures exposed to 3H-leucine (50µCi/ml) in the presence of the protein synthesis inhibitor cyclohexamide (1µg/ml, Sigma).

AChE activity assay

AChE activity was measured in cultured DRG neurons plated in 6-well plates at a density of 350 cells/mm2 using the Ellman assay (Ellman et al., 1961) as previously described (Howard et al., 2005). Tetraisopropyl pyrophosphoramide (100µM, Sigma) was included to inhibit pseudocholinesterase, and AChE activity measured in triplicate wells was normalized to protein concentration as determined using the BCA™ Protein Assay Kit (Pierce, Rockford, IL). Data are expressed as µmol substrate formed/min/mg protein.

Statistical analyses

Data were analyzed by ANOVA for treatment effects. If significant effects were identified (p < 0.05), post-hoc analyses were performed using the Newman-Keuls Multiple Comparison Test. Data are expressed as the mean ± SEM and p values indicated in the figure legend are from post hoc analyses.

RESULTS

When cultured on a poly-D-lysine substrate in serum-free medium, dissociated E15 rat DRG neurons extend 1–2 axons during the first 24h in vitro (Fig. 1A). This morphology is altered in cultures exposed to CPF (Fig. 1B). Morphometric analyses indicated that CPF did not alter the number of axons extended by DRG neurons (Fig. 1C), but did significantly decrease axonal length (Fig. 1D). The threshold concentration for this inhibitory effect of CPF on axonal growth was ≤ 0.001µM. Since CPF can be rapidly metabolized to the oxon metabolite in vivo, we next analyzed the effect of CPFO on axonal growth. CPFO is several orders of magnitude more potent than CPF in inhibiting AChE in cultured brain cell aggregates (Monnet-Tschudi et al., 2000), therefore, we used concentrations of CPFO that were several orders of magnitude lower than the CPF concentrations used in axonal growth experiments. Similar to observations of cultures treated with CPF, a 24h exposure to CPFO had no effect on axon number (Fig. 1C), but significantly decreased axonal length at a threshold concentration between 0.001 and 0.01nM (Fig. 1D).

Figure 1. Chlorpyrifos (CPF) and its oxon metabolite (CPFO) inhibit axonal growth in primary cultures of embryonic rat dorsal root ganglia (DRG) neurons.

Figure 1

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DRG neurons were treated with varying concentrations of CPF or CPFO during the first 24h in vitro, then fixed and immunostained for the neuronal antigen PGP9.5. Representative fluorescence micrographs of DRG neurons grown in the absence (A) or presence (B) of CPF (0.1µM) demonstrate that relative to vehicle controls, neurons treated with CPF exhibit shorter axons. CPF and CPFO did not affect the number of axons per neuron (C), but significantly did decrease axon length (D). V=vehicle control. *p<0.05 (n = 45). Bar, 25µm.

To rule out the possibility that inhibitory effects of CPF and CPFO on axonal growth are due to general cytotoxicity, the effect of these OPs on cell viability was assessed in cultured DRG neurons by determining the uptake of calcein AM and propidium iodide (Vaughan et al., 1995). Exposure to the same range of CPF and CPFO concentrations shown to decrease axonal growth had no effect on the viability of DRG neurons relative to vehicle controls (Fig. 2A). Previous reports demonstrated that CPF inhibits RNA and protein synthesis coincident with decreased neurite extension in differentiated PC12 cells (Song et al., 1998), suggesting that the inhibitory effects of OPs on axonal growth may occur secondary to decreased protein synthesis. To address this possibility, we determined whether CPF inhibited protein synthesis in cultured DRG neurons as measured by incorporation of 3H-leucine. CPF had no effect on 3H-leucine uptake at concentrations that significantly inhibited axonal growth in cultured DRG neurons (Fig. 2B).

Figure 2. Pesticide effects on cell viability, protein synthesis and AChE enzymatic activity.

Figure 2

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Treatment for 24h with CPF and CPFO at concentrations that inhibit axonal growth did not alter cell viability (A) as determined by uptake of calcein AM and propidium iodide (n = 6 fields) or inhibit protein synthesis (B) as determined up uptake of 3H-leucine (n = 3 cultures). (C) AChE enzymatic activity was inhibited by concentrations of CPF ≥ 0.1µM and CPFO ≥ 0.1nM but lower concentrations of CPF and CPFO that inhibit axonal growth did not significantly alter AChE enzymatic activity (n = 3 cultures). V= vehicle control. ** p<0.01.

Acetylcholine has been shown to inhibit neurite outgrowth when applied directly to cultured neurons (Lauder and Schambra, 1999), therefore, the inhibitory effect of CPF and CPFO on axonal growth in cultured DRG neurons could reflect increased levels of acetylcholine as a result of AChE inhibition. This seems unlikely since cultured DRG neurons are not cholinergic (Schoenen et al., 1989; Chauvet et al., 1995). However, because AChE inhibition is widely used as a biomarker of exposure to toxic levels of OP pesticides, we measured AChE activity in DRG cultures treated for 24h with varying concentrations of CPF, CPFO or TCP to determine if OP effects on neuronal morphogenesis correlate with AChE inhibition. Both CPF and CPFO inhibited AChE activity significantly at concentrations ≥0.1µM or 0.1nM, respectively (Fig. 2C). However, these OPs did not inhibit AChE activity at lower concentrations (Fig. 2C) that were previously shown to significantly decrease axonal growth (Fig. 1C, 1D).

The observation that CPF and CPFO inhibit axonal growth in cultured DRG neurons at concentrations that do not inhibit the enzymatic activity of AChE suggests two possibilities: (1) OPs disrupt the morphogenic activity of AChE independent of effects on the enzymatic activity of AChE; or (2) the inhibitory effect of CPF and CPFO on axonal growth are mediated by interactions with molecules other than AChE. To distinguish between these possibilities, we compared the effects of CPF and CPFO on axonal growth in DRG neurons from AChE null (AChE−/−) versus congenic wildtype (AChE+/+) mice. If AChE is the critical molecular target, then these OPs should inhibit axonal growth in AChE+/+ DRG neurons but not in AChE−/− DRG neurons. To establish pure cultures of AChE+/+ or AChE−/− DRG neurons, we pooled DRG from pups of the same AChE genotype as determined by the level of AChE activity in brain homogenates obtained from individual pups at the time of dissection. Wildtype (AChE+/+) animals express the highest levels of AChE activity; levels of AChE activity in heterozygous (AChE+/−) animals is approximately 50% of that measured in wildtype animals, and nullizygous (AChE−/−) animals express little to no AChE activity (Fig. 3A). Genotypes of all pups used for dissection were subsequently verified by PCR, and if PCR analyses indicated that AChE activity had not correctly predicted AChE genotype, cultures from that dissection were not used for subsequent experiments.

Figure 3. CPF and CPFO inhibit axonal growth in DRG neurons derived from AChE wildtype (AChE+/+), but not AChE nullizygous (AChE−/−) mice.

Figure 3

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(A) AChE enzymatic activity in brain homogenates from E14 pups as determined using the Ellman assay is predictive of AChE genotype as determined by PCR analyses (n = 6). (B) Representative fluorescence micrographs of DRG neurons derived from AChE+/+ or AChE−/− mice grown in the absence or presence of CPF (0.1µM) during the first 24h in vitro and then fixed and immunostained for the neuronal antigen PGP.9.5. (C, D) Quantification of axonal length indicate that in the absence of OPs, AChE−/− DRG neurons exhibited significantly attenuated axonal growth relative to AChE+/+ neurons. Exposure to either CPF (C) or CPFO (D) significantly decreased axonal growth in AChE+/+ neurons but had no effect on axonal growth in AChE−/− neurons. V=vehicle control, n = 50. *Significantly different from vehicle control of same AChE genotype at p<0.05. #Significantly different from wildtype vehicle control at p<0.05. Bar, 25µm.

In the absence of OPs, AChE−/− DRG neurons exhibited decreased axonal growth relative to AChE+/+ DRG neurons, confirming that AChE influences axonogenesis in this neuronal cell type. Consistent with observations of DRG neurons cultured from E15 rat pups, axonal length of DRG neurons cultured from AChE+/+ E14 mouse pups was significantly decreased following a 24h exposure to CPF (Fig. 3C). In contrast, a 24h exposure to the same concentration range of CPF had no effect on axonal length in AChE−/− DRG neurons (Fig.3C). Similarly, CPFO inhibited axonal growth in AChE+/+ DRG neurons, but not in AChE−/− DRG neurons (Fig. 3D).

These observations raised several questions. First, do the differential responses of AChE−/−versus AChE+/+ DRG neurons specifically reflect deletion of the AChE gene or are they due to non-target effects? Second, are the inhibitory effects of CPF and CPFO on axonal growth mediated by interactions with Ser203, which is the serine residue of the catalytic triad phosphorylated by OPs to inhibit AChE enzymatic activity (Mileson et al., 1998)? To address these questions, we determined the effect of CPF on axonal growth in AChE−/− DRG neurons transfected with either full-length wildtype AChE or mutant AChE rendered catalytically inactive by mutating Ser203 to Ala203. Characterization of the expression vectors encoding wildtype AChE (pAChE) and mutant AChE (pAChEΔ) in both COS7 cells (transfection efficiency of 60–80%) and DRG neurons (transfection efficiency of 10–20%) demonstrated that transfection with pAChE significantly increased AChE activity whereas transfection with pAChEΔ had no effect on AChE activity (Fig. 4A). RT-PCR analyses (Fig. 4B) followed by sequencing of the RNA amplified by AChE-specific primers (data not shown) confirmed that AChEΔ was expressed in both COS7 cells and DRG neurons transfected with pAChEΔ.

Figure 4. Mutation of Ser203 to Ala203 disrupts the enzymatic activity of AChE.

Figure 4

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(A) Analysis of AChE enzymatic activity using the Ellman assay confirms that pAChEΔ construct is catalytically inactive when expressed in either COS7 cells or AChE−/− DRG neurons. *p<0.05, **p<0.01 (n = 3 cultures). (B) RT-PCR analyses using primers that specifically recognized pAChEΔ confirmed that this construct was expressed in COS7 cells and in AChE−/− DRG neurons following transfection.

Transfection of AChE−/− DRG neurons with pGFP alone did not alter axonal growth of these neurons in the absence or presence of CPF (Fig. 5A, 5C). However, co-transfection with pGFP and pAChE restored the wildtype phenotype to AChE−/− DRG neurons, evident as both significantly increased axonal length in the absence of CPF, and significant inhibition of axonal growth in the presence of CPF (Fig. 5B, 5C). Interestingly, co-transfection of AChE−/− DRG neurons with pAChEΔ did not alter the phenotype of these neurons with respect to either basal axonal growth or responsiveness to the axon inhibitory effects of CPF (Fig. 5C).

Figure 5. Expression of wildtype but not mutant AChE restores wildtype phenotype to AChE−/− DRG neurons.

Figure 5

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Representative fluorescence micrographs of AChE−/− DRG neurons transfected with pGFP alone (A) or co-transfected with pGFP and pAChE (B). Neurons were treated with CPF for 24h starting the day after transfection. Transfected neurons were identified by GFP expression (green fluorescence) and axons delineated by immunostaining for phosphorylated neurofilaments (red fluorescence). (C) Transfection of AChE−/− DRG neurons with wildtype AChE (pAChE) caused a significant increase in axon length and restored responsiveness to the axon inhibitory effects of CPF. Transfection of AChE−/−DRG neurons with mutant AChE (pAChEΔ) did not alter axonal growth relative to control cultures transfected with GFP alone in the absence or presence of CPF. *Significantly different from vehicle control transfected with pAChE at p<0.05,***p<0.001. # # Significantly different from vehicle control transfected with pGFP alone at p<0.01. Bar, 50 µm.

DISCUSSION

Developmental exposure to OPs especially CPF, at levels that do not elicit signs or symptoms of systemic toxicity or inhibit AChE enzymatic activity have been linked to neurobehavioral deficits in animal models (Jett et al., 2001; Levin et al., 2001; Levin et al., 2002) and, more recently, in children (Rohlman et al., 2005; Kofman et al., 2006; Eskenazi et al., 2007; Lizardi et al., 2007). Consistent with these observations, the studies reported herein identify axonal growth as a neurodevelopmental event targeted by OPs independent of decreased AChE enzymatic activity. However, our data indicate that the axon inhibitory effect of the OPs CPF and CPFO does require neuronal expression of AChE, suggesting that OPs inhibit axonal growth by interfering with the morphogenic activity of AChE.

We demonstrate in this study that in DRG cultures with normal AChE expression, CPF and CPFO did not change the number of axons but significantly decreased the length of the axonal plexus extended by individual neurons, and this effect was observed at concentrations that neither impaired cell viability nor inhibited the enzymatic activity of AChE. These observations are consistent with our previous report that CPF and CPFO, but not 3,5,6-trichloro-2-pyridinol (TCP), decrease the rate of axonal growth in primary cultures of sympathetic neurons (Howard et al., 2005), and with studies published by other laboratories demonstrating that these and other OPs inhibit neurite outgrowth in neural cell lines (Li and Casida, 1998; Song et al., 1998; Das and Barone, 1999; Sachana et al., 2001; Hargreaves et al., 2006). Similar to the current study, the dysmorphogenic effects observed in cultured sympathetic neurons and neural cell lines occur independent of cytotoxicity and/or inhibition of AChE enzymatic activity. Considered in aggregate, these studies provide strong support for the argument that OPs specifically target mechanisms of axonal growth.

Axonal growth is influenced by diverse environmental cues that activate different proximal signaling pathways but ultimately converge upon common effector mechanisms that regulate membrane and cytoskeleton dynamics (Dent and Gertler, 2003; Huber et al., 2003; Gallo and Letourneau, 2004). OPs have been shown to modulate several of the factors involved in controlling axonal growth. For example, in experimental animal models, developmental exposures to CPF at doses that did not cause signs of overt toxicity or of cholinergic hyperstimulation were found to significantly alter proliferation and/or survival of neuronal and glial cells (Roy et al., 2004); decrease expression of neurotrophic factors, specifically, nerve growth factor (NGF) and reelin (Betancourt et al., 2006); and inhibit synthesis of nucleic acids and proteins (Campbell et al., 1997; Johnson et al., 1998; Garcia et al., 2001). However, it seems unlikely that these effects of OPs are responsible for the inhibition of axonal growth in cultured DRG neurons. For example, the DRG neurons used for these studies were post-mitotic at the time of dissociation (Kleitman et al., 1998), and CPF and CPFO treatments that decreased axonal length had no effect on cell viability, suggesting that OP effects on axonal growth were not the consequence of OP effects on neuronal and glial cell proliferation or survival. Our observations also argue against a role for NGF or reelin since DRG neurons were cultured in medium supplemented with maximally effective concentrations of NGF, and deletion of AChE was sufficient to decrease axonal growth to levels comparable to those observed in wildtype neurons treated with OPs, an effect that was completely reversed by transfection with full length AChE cDNA. Our previous report that inhibition of RNA or protein synthesis does not influence axonal growth in cultured sympathetic neurons during the first 24h in vitro (Lein and Higgins, 1991) suggests that OP effects on axonal growth are not mediated by inhibition of macromolecule synthesis. This conclusion is substantiated by our finding in this study that CPF did not inhibit protein synthesis in cultured DRG neurons at concentrations that significantly decreased axonal length. The lack of effect of CPF on protein synthesis in cultured DRG neurons stands in contrast to previous reports that CPF inhibits macromolecule synthesis in PC12 and C6 glioma cells (Garcia et al., 2001; Qiao et al., 2001). However, when these cell lines were grown in the absence of serum, CPF inhibited DNA and protein synthesis at concentrations ≥ 15µM, whereas the highest concentrations tested in our system was 10µM. Therefore, the discrepancy between these previous studies and our findings may reflect differences in the range of CPF concentrations used. Alternatively, the discrepancy may be due to inherent differences between primary neuronal cell cultures and transformed cell lines (Lein et al., 2005).

Disruption of cytoskeletal proteins has been proposed as a mechanism to explain inhibition of neurite outgrowth in cell lines exposed to OPs (Sachana et al., 2001; Hargreaves et al., 2006; Flaskos et al., 2007). However, it has yet to be established whether OPs directly target cytoskeletal proteins, which then results in decreased neurite outgrowth, or whether decreased expression of cytoskeletal proteins is a consequence of decreased neurite outgrowth. If OPs inhibit axonal growth by direct effects on the cytoskeleton, then culturing neurons on different substrates would not be expected to qualitatively change the axonal response to OPs. However, we have previously demonstrated that CPF significantly inhibits axonal growth in primary sympathetic neuronal cell cultures plated on poly-D-lysine but has no effect on axonal growth in sister cultures grown on laminin (Howard et al., 2005), suggesting that CPF targets signaling molecules upstream of the cytoskeleton. Data demonstrating that culturing neurons on laminin significantly downregulates AChE expression (Gupta and Bigbee, 1992; Howard et al., 2005), implicates AChE as a potential upstream signaling molecule targeted by OPs.

The first suggestion that AChE functions as a morphogen to promote axonal growth came from observations that, in situ, many neuronal cell types, even those that are neither cholinergic nor cholinoceptive, express AChE along axons and in axonal growth cones during periods of axonal outgrowth (Bigbee et al., 1999; Brimijoin and Koenigsberger, 1999). Subsequent functional studies demonstrated that antisense suppression of AChE decreased neurite outgrowth in PC12 cells, and this could be rescued by heterologous expression of the synaptic form of human AChE (Grifman et al., 1998). Similarly, transfection of neuroblastoma cells with antisense AChE decreased neurite outgrowth whereas transfection with sense AChE enhanced neurite outgrowth, and this latter effect was blocked by AChE antibodies (Koenigsberger et al., 1997). These results have been replicated in primary cultures of Xenopus motor neurons (Sternfeld et al., 1998) and rat DRG neurons (Bigbee et al., 2000). Consistent with these reports, we observed in this study that axonal growth in DRG neurons derived from AChE−/− mice is attenuated relative to that observed in DRG neurons derived from AChE+/+ mice. Interestingly, axonal lengths of AChE−/− DRG neurons were similar to those of AChE+/+ DRG neurons exposed to CPF or CPFO. These data not only confirmed that AChE influences axonal growth in cultured DRG neurons, but also suggested that AChE may be the critical molecular target for OP effects on axonal growth. More definitive support for this latter hypothesis was provided by our finding that relative to genotype-matched vehicle controls, CPF and CPFO inhibited axonal growth in AChE+/+ but not AChE−/− DRG neurons. The lack of axonal response to OPs was specifically due to deletion of AChE since transfection with full-length AChE cDNA fully restored the wildtype axonal phenotype to AChE−/− DRG neurons both in the absence and presence of CPF. These studies demonstrate that AChE is required for the axonal response to OPs. Since OPs inhibit axonal growth independent of effects on the enzymatic activity of AChE, these data strongly support the hypothesis that OPs interfere with the morphogenic activity of AChE.

Our data demonstrating that the OPs CPF and CPFO can independently modulate the morphogenic and enzymatic activities of AChE are consistent with several lines of experimental evidence suggesting that the enzymatic activity of AChE is not required for its morphogenic function. First, pharmacological inhibitors of cholinesterases have similarly been reported to block AChE-induced neurite outgrowth at concentrations that did not inhibit the enzymatic activity of AChE (Layer et al., 1993;Ling et al., 1995; Dupree and Bigbee, 1996; Koenigsberger et al., 1997; Chiappa and Brimijoin, 1998; Munoz et al., 1999). Interestingly, this effect was caused by pharmacological inhibitors that bind to the peripheral anionic site (PAS), but not by those that bind to the active site. Second, Xenopus spinal motor neurons transfected with mutated AChE cDNA encoding a nonfunctional catalytic domain exhibited neurite outgrowth comparable to that of neurons transfected with intact wildtype AChE mRNA, and both transfectants exhibited enhanced neurite outgrowth relative to nontransfected controls (Sternfeld et al., 1998). These studies not only demonstrate that AChE can function as an axonal morphogen, but also strongly suggest that separate structural domains mediate the morphogenic (PAS) and enzymatic (active or acylation site) activities of AChE.

OPs interact with the acylation site to phosphorylate the active site Ser203, resulting in inhibition of the enzymatic activity of AChE (Mileson et al., 1998). This well-characterized interaction is not necessarily inconsistent with the data indicating that Ops alter axonal growth independent of inhibition of AChE enzymatic activity in light of recent evidence demonstrating functionally significant conformational interactions between the PAS and the active site, and binding of various OPs, including CPF and CPFO, to not only the acylation site but also the PAS (De Ferrari et al., 2001; Kousba et al., 2004; Rosenfeld and Sultatos, 2006). This suggests at least two mechanisms by which CPF and CPFO might interfere with the morphogenic activity of AChE: (1) Binding of these OPs to active site Ser203 alters the morphogenic function of the PAS via conformational interactions; or (2) OPs bind directly to the PAS to interfere with morphogenic activity. In an attempt to distinguish between these possibilities, we determined whether mutating the active site Ser203 to Ala203 altered the axonal response to OPs. Evidence that this mutation attenuated the inhibitory effects of OPs on axonal growth would support the former; whereas evidence suggesting that the mutation did not alter axonal effects of OPs would argue for the latter. Enzymatic analyses of COS7 cells and AChE−/− DRG neurons transfected with pAChEΔ, the plasmid carrying this mutant form of AChE, confirmed that this construct encoded catalytically inactive AChE. However, subsequent morphometric analyses of AChE−/− DRG neurons expressing this catalytically inactive AChE construct indicated that this point mutation also disrupted the morphogenic activity of AChE, as evidenced by the failure of this construct to restore the wildtype phenotype with respect to basal levels of axonal growth in the absence of OPs. Sequence analysis of pAChE.Δ confirmed that the Ser203 to Ala203 mutation was the only mutation introduced into the full-length wildtype AChE cDNA, and RT-PCR confirmed that the mutant construct was expressed. Considered in aggregate, these data suggest that Ser203 is necessary for the morphogenic activity of AChE. This was an unexpected finding based on previous studies demonstrating that expression of a catalytically inactive form of AChE enhanced neurite outgrowth in Xenopus spinal motor neurons to the same extent as a catalytically active form of AChE (Sternfeld et al., 1998). While the reason(s) for the discrepancy between this study and our current findings are not known, technical differences between the studies suggest several possibilities. First, Sternfeld and colleagues used an insert-disrupted form of the enzyme in which an in-frame sequence of 21 nucleotides was inserted six bases downstream of the codon for Ser203; thus, it is possible that while this insertion was sufficient to disrupt the enzymatic capacity of AChE, it had minimal impact on the morphogenic activity because Ser203 was still present. Second, it has been proposed that the morphogenic function of AChE is mediated by homophilic interactions between AChE molecules (Small et al., 1996; Brimijoin and Koenigsberger, 1999; Grisaru et al., 1999). Unlike the current study in which the mutant AChE construct was expressed in cells totally devoid of AChE, Sternfeld and colleagues expressed their insertion-inactivated AChE construct in neuronal cells expressing endogenous levels of wildtype AChE; thus, it is possible that the interactions of the mutant AChE with endogenous wildtype AChE was sufficient to compensate for the decreased morphogenic activity in the mutant protein. Third, the apparent discrepancy may reflect species- (Xenopus versus mouse) or neuronal cell type- (spinal versus DRG neuron) differences. While the specific role of Ser203 in the morphogenic activity of AChE, as well as the molecular mechanism by which Ops alter this function of the enzyme remain to be determined, it seems clear from this and previous studies (Das and Barone, 1999; Howard et al., 2005), that the morphogenic activity of AChE is more sensitive to disruption by OPs than the enzymatic activity of AChE.

The relationship of OP effects on axonal growth to the neurobehavioral deficits observed in animals or children exposed developmentally to OPs remains to be determined. However, there is an extensive literature demonstrating that the functional properties of the vertebrate nervous system are determined by the pattern of neural connections formed during development, and that even subtle disruption of the temporal or spatial control of axonal growth can result in functional deficits (Berger-Sweeney and Hohmann, 1997; Cremer et al., 1997; Cremer et al., 1998; Barone et al., 2000; Rice and Barone, 2000). Our data demonstrate that CPF and CPFO inhibit axonal growth, and suggest that this effect is mediated, at least in part, by interference with the morphogenic activity of AChE. These findings support the novel hypothesis that age-related differences in the function of AChE contribute to the age-related vulnerability to OP neurotoxicity. While the molecular mechanisms by which OPs modulate the morphogenic function of AChE independent of effects on the enzymatic function of AChE have yet to be defined, our data suggest that altered neuronal morphogenesis may be an important mechanism mediating the developmental neurotoxicity of OPs, and that the use of AChE enzymatic activity as a biomarker of OP neurotoxicity may not be relevant in assessing children’s vulnerability to these neurotoxicants.

ACKNOWLEDGEMENTS

This work was supported by NIH grants R21 ES011771 (PJL) and T32 ES007141 (ASH) and by startup funds provided by the Center for Research on Occupational and Environmental Toxicology (CROET), Oregon Health & Science University (PJL). These sponsors were not involved in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.

Footnotes

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CONFLICT OF INTEREST STATEMENT None of the authors have a conflict of interest.

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