9MOLECULAR TOXICOLOGY OF NEUROPATHYTARGET ESTERASE

YI-JUN WU

Laboratory of Molecular Toxicology, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology,Chinese Academy of Sciences, Datunlu Road, Beijing 100101, China

PING-AN CHANG

Key Laboratory of Molecular Biology, College of Bio-information, Chongqing University of Posts and Telecommunications, Chongqing,400065, China

9.1 Introduction 109

9.2 Molecular Biology of Neuropathy Target Esterase 1109.2.1 Discovery of Neuropathy Target Esterase 1109.2.2 Biochemistry of NTE 1109.2.3 Molecular Structure of NTE 1119.2.4 Distribution and Function of NTE 1129.2.5 Regulation of NTE Expression 113

9.3 Molecular Toxicology of Neuropathy Target Esterase 1149.3.1 NTE and OPIDN 114

9.3.2 The Pathway of NTE Aging 1149.3.3 Mechanisms of OPIDN Related to NTE 1159.3.4 Application of NTE in the Prediction

of Toxicity 116

9.4 Prospects of Molecular Toxicology of NTE 117

9.5 Conclusions 118

Acknowledgment 118

References 118

9.1 INTRODUCTION

Organophosphorus compounds (OPs) are used commerciallyas pesticides, including insecticides and herbicides, in agri-culture; as additives, including plasticizers, flame retardants,and lubricants in industry; as therapeutic agents in medicine;and as nerve agents in warfare (Gupta, 2006). Most OPs arehighly toxic. They have caused serious damage to the eco-logical environment and human health, which has been ofconcern to environmental toxicologists.

Two main types of poisoning effects can be observed inhuman and susceptible animals after exposure to OPs. Oneof them is acute toxicity, because of the inhibition of acetyl-cholinesterase (AChE) by the OP and then the accumulationof acetylcholine in the synapses leading to blocking of con-duction in nerve signals. The other is delayed neurotoxicity,so-called organophosphate-induced delayed neurotoxicity

(OPIDN) after a single or multiple exposures to some OPs,which is also called organophosphate-induced delayed poly-neuropathy (OPIDP), with characteristics of paresthesia,muscle pain, limb weakness, and even paralysis (Johnson,1993). In addition, some OPs can cause intermediate syn-drome (IMS), which appears between day one and day fourafter the acute poisoning. Symptoms of IMS are myastheniaand dyspnea, and it has a high mortality (Senanayake andKaralliedde, 1987). For further details on the mechanismsof IMS, readers are referred to De Bleecker (2006).

The earliest case of OPIDN was reported in 1930. Tens ofthousands of Americans suffered from poisoning, with signsof ataxia and paralysis after drinking alcoholic extract ofJamaican ginger that was contaminated with tri-o-cresylphosphate (TOCP). Similar cases of TOCP poisoning alsooccurred in Mexico, Sri Lanka, and China. The surveyshowed that besides TOCP, warfare agents such as sarin,

Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Edited by Tetsuo Satoh and Ramesh C. GuptaCopyright # 2010 John Wiley & Sons, Inc.

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and organophosphorus insecticides such as parathion, mala-thion, and dichlorvos, can induce delayed neurotoxicity invarying degrees in susceptible animals. Hens have becomethe animal model for OPIDN study. All OPs that inducedelayed neurotoxicity in humans can induce the same delayedneurotoxicity in hens. In addition, the signs, onset of symp-toms, and histopathology of the affected hens are very similarto those of humans. In addition the chicken as a laboratoryanimal is easy to obtain and easily treated.

More than 70 years have passed since the first case ofOPIDN was found and some progress has been made inresearch on OPIDN; however, the exact mechanism stillremains unclear. The organophosphorus insecticides arebeing replaced gradually in developed countries by low-toxicity pesticides, but many OPs are widely used in develop-ing countries. Several OP nerve agents, including sarin,soman, tabun, VX, etc., are still used as deterrent weapons.Therefore, it is of practical significance to study the mechan-isms of OPIDN.

9.2 MOLECULAR BIOLOGY OF NEUROPATHYTARGET ESTERASE

9.2.1 Discovery of Neuropathy Target Esterase

Researchers have been looking for a potential target since1954 when Aldridge proposed that the phosphorylation ofesterase is the primary event of OPIDN. Martin K. Johnsonfound a protein with esterase activity in chicken brain hom-ogenates that was inhibited selectively by OPs. This esterase,which was then generally called neuropathy target esterase,was named neurotoxic esterase (NTE) by Johnson (1969,1974). However, all OPs that can covalently inhibit NTEare not neuropathic ones. Johnson found that some OPscannot induce delayed neurotoxic signs in hens even if theyinhibited the NTE activity. Therefore, Johnson speculatedthat it is necessary for the OPIDN during which the NTEactivity was inhibited (phosphorylation) by the neuropathicOPs and then underwent the next step, that is, “aging”; theinhibited NTE cannot be reactivated (Johnson, 1974).Johnson also speculated that aging is a cleavage of R groupfrom the OP part of the phosphorylated esterase and agroup with negative charge linked to the active site of theenzyme.

NTE was thought to be the primary target of neuropathicOPs for OPIDN. The prerequisite of OPIDN was that NTEmust be inhibited to a level of less than 70% of the normalNTE activity in hen, while the sufficient condition forOPIDN is the aging of the inhibited NTE and the enzymecannot be reactivated. Based on these experimental results,Johnson suggested that the inhibition and aging of theenzyme was the early biochemical changes of NTE. NTEaging probably affected some characteristics of the

nonenzyme features of the protein or changed the environ-ment of the reaction and then induced the delayed neurotoxi-city through some unknown process (Johnson, 1974).

9.2.2 Biochemistry of NTE

Since NTE was found to be a primary target for initiation ofOPIDN, separation and purification of NTE became the focusfor biochemists and toxicologists to study the characteristicsof the enzyme. Early studies revealed that NTE is a macro-molecular enzyme that is different from the serine esterasefamily. SDS-PAGE analysis showed that the molecularweight of NTE is about 155 kDa, which is at least twotimes the molecular weight of the known serine esterases(Williams and Johnson, 1981). The molecular weight of thecatalytic part of NTE on the chick brain membrane is 105kDa (Carrington et al., 1985). NTE is a transmembraneprotein because it was found to hydrolyze phenyl valerateonly in the presence of membrane lipids. Therefore, the con-centration and type of the detergents used to resolve the cat-alytic NTE from the membrane of brain microsomes shouldbe selected carefully. The addition of phospholipid was help-ful to maintain the activity of the enzyme (Pope and Padilla,1989). The native SDS-PAGE analysis revealed that the mol-ecular weight of the oligocomplex of NTE, lipid, and thedetergent was 850 kDa (Thomas, 1990). There are multipletypes of AChE oligomers and some of them can bind to themembrane by the glycolipid at the end of the chain; however,none of the cholinesterase family members with catalyticactivity has the polypeptide chains with a transmembranestructure. There is a particular reaction of NTE with theOPs; the inhibited NTE must undergo a so-called “aging”reaction. The nature of the aging is cleavage of the OP partfrom the phosphorylated esterase and a substituted groupwith negative charge linked to the active site. The separatedR group is then transferred to other residues of NTE ratherthan into the reaction solution as occurs with other serineesterases (Williams, 1983). This process is usually finishedwithin minutes, which is much faster than other serineesterases. This phenomenon indicates that the assignmentof amino acids to the catalytic site is probably differentfrom that of other serine esterases.

The characteristics of the enzyme will only be knownwhen the NTE protein is obtained with catalytic activity.However, it is very difficult to separate and purify NTEbecause the protein is a transmembrane macromoleculewith serine esterase activity. Work on isolation and purifi-cation of NTE did not make substantial progress until 1994.At that time, a study demonstrated that S9B, a kind of OPcoupled to avidin, was labeled and then bound to NTElocated on microsome membranes; the membrane proteinwas then resolved into 0.15% SDS. The NTE bound toavidin can be absorbed to an affinity chromatographiccolumn. This method was the base of further purification of

110 MOLECULAR TOXICOLOGY OF NEUROPATHY TARGET ESTERASE

NTE by affinity chromatography (Glynn et al., 1994). In1998, Lush et al. reported that the NTE labeled with S9Bwas hydrolyzed by protease V8 and then the isolated frag-ment was sequenced in its N-terminal. They found that thesequence of one of the peptide fragments was the same asthat of an express sequence tag (EST) in humans. Theyused this EST to screen the human fetal cDNA library andcloned the human NTE cDNA sequence (Lush et al., 1998).

NTE cDNA sequence cloning is a key step forward tounderstanding the NTE feature. It was deduced from thecDNA sequences that NTE is a single chain protein with1327 amino acids. It is a shortcut pathway to study NTE bythe in vitro expression method after obtaining the NTEcDNA sequence. The biochemical test showed that theserine residue of the catalytic S9B-NTE lay in amino acids955-1033. More attention was focused on the Ser966 becauseit is located in the motif of G-X-S-X-G, the marker sequenceof serine esterase and serine protease (Lush et al., 1998). Arecombined peptide, which can hydrolyze phenyl valerateand is sensitive to OPs, was found to have at least thesequences of amino acids 727-1216 in Escherichia coli.This recombined peptide sequence was called NTE esterasedomain (NEST; Atkins and Glynn, 2000). It was alsofound that only when binding to membrane is NEST cataly-tically active, which verified that NTE is a kind of membraneprotein. The results of site mutation research and patatindomain model analysis indicate that the amino acids withinthe active site of NTE are Ser966 and Asp1086, althoughAsp960 is also a key amino acid for NTE catalytic activity(Atkins and Glynn, 2000; Wijeyesakere et al., 2007), whichis different from that of serine protease. Amino acids withinthe active center of the latter are usually Ser, His, and Aspor Glu. In addition, Asp1044 and Asp1004 were assumed tobe the binding sites for transferring the R group of OPduring aging (Atkins and Glynn, 2000). The in vitroexpression test showed that NTE is catalytically active evenin its monomer without need of formation of oligomer orpolymer (Atkins et al., 2002).

By comparing the amino acid sequence of NTE in theactive site regions with that of calcium-independent phospho-lipase A2 (iPLA2), it was found that they have similarsequences of the active sites. Therefore, it was speculatedthat NTE can hydrolyze membrane lipid also. It was foundthat NEST expressed in yeast can react with membranelipids and hydrolyze 1-palmitoyl-lysophosphatidylcholineat the maximum reaction rate. Therefore, NTE was con-sidered as a kind of lysophospholipase (van Tienhovenet al., 2002). NTE knock-out mice were used to study theNTE function and the inhibition rate of NTE in the brainwas found to be very close to that of lysophospholipase,giving a good correlation. Many NTE inhibitors were usedto study the enzyme in vitro and in vivo and there is a similarinhibition level for NTE and lysophospholipase. Therefore,it was confirmed that NTE is actually a type of

lysophospholipase, which hydrolyzes lysophosphatidylcho-line in the body (Quistad et al., 2003). In yeast, the codingproduct of open reading frame YML059c is very similar tothat of NTE, which is named NTE1 (Murray andMcMaster, 2005). NTE1 catalyzes the deacylation reactionto produce glycerophosphocholine (GPC) and fatty acids.In the absence of YML059c, no intracellular accumulationof GPC could be observed, which suggested that NTE1 wasthe sole protein for catalyzing the deacylation reaction oflysolecithin in the yeast. Overexpression of NTE in COS7and HeLa cells led to increase in GPC levels, which can beinhibited by reduction of the choline absorbtion and CDP-choline synthesis. However, the GPC levels were diminishedby the reduction of endogenous NTE activity mediated byeither RNA interference or OP treatment, which confirmedthat NTE catalyzes the deacylation of phosphatidylcholinethrough the pathway of CDP-choline synthesis (Zaccheoet al., 2004). In addition to cell experiments, animal exper-iments also found that NTE protein in mice can rescue thedefects that arise in Swiss cheese protein (SWS) genemutant flies, suggesting that the function of fly SWS proteinis similar to that of mouse NTE. The levels of phosphatidyl-choline in the brain of a SWS mutant fly or in the NTE over-expression fly are higher or lower than that in the wild type,respectively, which confirmed that physiological substanceof NTE in the body is phosphatidylcholine (Muhlig-Versenet al., 2005). It is essential for understanding of NTE thatthe enzyme is a phospholipase B catalyzing phosphatidyl-choline synthesized by CDP-choline.

9.2.3 Molecular Structure of NTE

NTE is a single-chain protein of 1327 amino acids, which wasdeduced from a cDNA sequence of human NTE. Mouse brainNTE also encodes 1327 amino acids, which is 96% identicalto human NTE (Moser et al., 2000). The human NTE gene islocated on the region of chromosome 19p13.3–p13.2, neigh-boring the region of mucolipidosis type IV gene MCOLN1(Acierno et al., 2001). The length of the human NTE geneis 26.4 kb, with 35 exons. After splicing or transcription, itbecomes a cDNA sequence of 4.3 kb encoding 1327 aminoacids (Acierno et al., 2001). Mouse NTE is 29 kb, also with35 exons. NTE genes from these two species share highsequence identity with regions of the introns and exons inthe genomics (Winrow et al., 2003).

Analysis of the protein domain and the transmembranesequences revealed that there is a transmembrane domainwithin amino acids 7 to 31 of the N-terminal in which thereare three cyclic nucleotide binding domains (amino acids163 to 262, 480 to 573, and 579 to 689), and one patatindomain (amino acids 939 to 1099) in the C-terminal(Fig. 9.1). Comparison of amino acid sequences showedthat there is only one protein with 61% identity in NTE inGenbank, which was called NTE-related esterase (NRE); it

9.2 MOLECULAR BIOLOGY OF NEUROPATHY TARGET ESTERASE 111

has no high degree of homology to the known serine esterasesand serine proteases (Chang et al., 2007). By comparison ofthe sequences of the protein from Genbank, NTE was foundto have 41% homology to fly SWS. In addition, NTE hassome degree of identity with the unknown proteins fromnematode, yeast, and other animals, especially in the regionclose to the amino acids 200 of the C-terminal; it is in highhomology to that of the proteins from bacteria, yeast, nema-tode, and fruit fly. Almost all regions in the regulatorydomain have similar sequences to that of protein kinase A(PKA) regulatory subunits, suggesting that these proteinsmay constitute a novel protein family. Due to the particularrelationship of NTE in the protein configuration and evol-ution, NTE and NRE in the ten patatin-containing phos-pholipase families constitute a novel subfamily, whichwas named PNPLA6 and PNPLA7, respectively (Wilsonet al., 2006).

Topology study of NTE revealed that there is only onetransmembrane domain (9 to 31 amino acids) located at theN-terminal part, through which NTE is anchored to the endo-plasmic reticulum (ER). Most of the rest of NTE is located inthe cytoplasm. The N-terminal and C-terminal can react withthe membrane of ER that faces the cytoplasm (Li et al., 2003).By expression of various fragments of NTE in the COS7cells, it was found that NEST has NTE activity and canbind to the ER membrane. Neither the transmembranedomain nor the regulatory domain of the N-terminal is essen-tial for NTE catalytic activity but this activity requires mem-brane association, to which the transmembrane, regulatory,and catalytic domains all contribute (Li et al., 2003).Bioinformatics analysis showed that the NTE patatindomain can be associated with the ER membrane, but itcannot be considered an integral membrane domain. Theresults of the experiments described above, along with analy-sis of a 3-D model of the patatin domain, led to a three-dimensional molecular model of NTE proposed byWijeyesakere et al. (2007; Fig. 9.2).

9.2.4 Distribution and Function of NTE

The function of NTE is closely related to its distribution in tis-sues and cells. Assay of NTE activity in tissue homogenatesshows that there is a high catalytic activity in the brain;

however, there is comparatively lower activity in the spinalcord and peripheral nerves. The ratio of NTE specific activityis 1.0 :0.2 :0.04 in the brain, spinal cord, and sciatic nerve,respectively. However, there are also relatively high activitiesof NTE in non-neuronal cells and tissues, such as testicles,kidneys, and lymphocytes. Immunohistochemical analysisindicates that NTE protein is expressed during the earlystage of embryonic neural development in mice and exhibitstemporal-spatial characteristics with the embryo development(Moser et al., 2000). In hen neural systems, NTE was foundonly in neurons but not in glia. Besides the nucleus, theneuron body and proximal part of the axons were also stainedby the specific antibody-dye (Glynn et al., 1998), which wasthen confirmed by autoradiography (Kamijima and Casida,1999). NTE was transported quickly in the axon of the sciaticnerves of hens (Glynn et al., 1998). Expression of NTE inCOS7 cells showed that this protein is localized on the mem-brane of ER but not on the cell plasma membrane(Akassoglou et al., 2004). Similarly, NTE1 or SWS wasalso localized on ER membrane in yeast or fly, respectively(Muhlig-Versen et al., 2005). It was thought previously thatNTE was expressed only in neurons but not in glia cells inmammalian brain; however, endogenous SWS was alsodetected in some glia of fly (Muhlig-Versen et al., 2005),which may be due to differences in sensitivity of the testmethods.

Figure 9.1 Protein domains of human neuropathy target esterase (NTE). From Wijeyesakere, S. J. et al. 2007. Protein J 26(3):165–172.Reproduced with permission.

Figure 9.2 Molecular model of NTE. TMD, transmembranedomain; CNP, cyclic nucleotide binding domain; PNTE, patatindomain of NTE; ER, endoplasmic reticulum. Adapted fromWijeyesakere, S. J. et al. 2007. Protein J 26(3):165–172.Modified with permission.

112 MOLECULAR TOXICOLOGY OF NEUROPATHY TARGET ESTERASE

The study of the relationship between NTE and OPIDN isthe basis of investigating NTE functions. It was suggestedthat NTE may be a type of receptor that mediates a signalpathway in the cell, based on the results of early studies onthe interactive action of NTE and OPs (Lotti and Moretto,1993). After the complete amino acid sequence of NTEbecame known, one started to speculate on NTE functionsbased on the function of other known proteins (actually, itis NTE function prediction). Human NTE share high identitywith fly SWS in the amino acid sequences; functions of NTEare probably similar to that of SWS. Neurons in wild-type flywere wrapped tightly with a layer of glia, while neurons in theSWS mutant fly were wrapped loosely with multiple layers ofglia and therefore blocked the interaction of neurons and gliacells, leading to apoptosis of these two types of cells andfinally vacuolation in most brain neural cells. SWS was con-sidered to be involved in the signal pathway of the inter-actions of neurons and glia during the neural developmentof fly. When wrapped with only one layer of glia, neuronssend signals to the adjacent glia cells to stop the wrapping pro-cess (Kretzschmar et al., 1997). In addition, SWS in wild-type fly possess the NTE-like activity that can hydrolyzephenyl valerate, while the mutant SWS has no activity. TheNTE-like activity in heterozygous SWS fly is higher than inthe mutant but lower than in the wild-type. It furthersuggested that NTE and SWS have similar functions. Thein situ hybridization study showed that NTE was expressedat the early stage of mouse embryo development (Moseret al., 2000). The function of NTE in vertebrates was probablyto mediate a signal pathway of glial wrappings during theinteractive process of the two types of cells (Glynn, 2000).

An often-used method to investigate gene function is toestablish a gene knockout animal model. The NTE geneknockout mice developed normally but displayed hyperactiv-ity signs, which can be induced by either the gene knockoutor OP treatment through NTE inhibition without requirementfor NTE aging (Winrow et al., 2003). However, mice with acomplete knockout of the NTE gene cannot develop normallyand die at embryonic day 9, which indicated that NTE isessential for embryo development. A further study showedthat the placenta development and the vascularization wereaffected. The histopathological analysis showed that NTE isrequired for the formation of the labyrinth layer and survivaland differentiation of secondary giant cells (Moser et al.,2004). A study found that the embryo of the mice lackingNTE can develop normally but exhibits neurodegenerativesigns. Absence of NTE resulted in disruption of the endoplas-mic reticulum, vacuolation of nerve cell bodies, and abnormalreticular aggregates. The neurodegeneration in the nervesystem lacking NTE was due to the neuronal vacuolizationand neuronal cell loss (Akassoglou et al., 2004). Age-relatedneurodegeneration can be found in SWS mutant fly. Theapoptosis and the multilayer glia wrapping neurons wereobserved in the central nerve system; however, specific

expression of fly SWS or mouse NTE can rescue the pheno-type of the wild-type flies (Muhlig-Versen et al., 2005).Overexpression of SWS also caused neurodegeneration anddeath of neurons and glia in fly (Muhlig-Versen et al.,2005). Therefore, SWS/NTE plays a very important role inthe embryonic and nervous development.

There is some understanding of the physiological functionof NTE in the body, but the roles of NTE in specific cellsremain unidentified. Studies using antisense RNA andRNA interference techniques show that NTE is not requiredfor the differentiation of human SK-N-SH cells (Changet al., 2005a, 2005b) although the expression of NEST inthe cells can promote the differentiation (Chang et al.,2005a). NTE overexpression can inhibit the proliferationnot only of non-neuronal COS7 cells but also human neuro-blastoma SH-SY-5Y cells and block the cycle at the G2/Mstage in COS7 cells (Chang et al., 2006). NTE is not essentialfor neurite initiation and elongation of mouse embryostem cells but it can affect the neurite outgrowth (Li et al.,2005). All the above results indicate that NTE plays a rolein the regulation of cell proliferation and differentiation.Considering the fact that NTE is phospholipase B and loca-lized on the membrane of ER, it is speculated that NTE regu-lates the levels of lysophosphocholine/phosphocholine andplays a role in the regulation of cell membrane formationand cell cycles.

9.2.5 Regulation of NTE Expression

NTE was divided into two parts based on the domains in themolecular structure. The N-terminal is the regulatory partcontaining three domains that are similar to the regulatorysubunits of PKA, and the C-terminal is the catalytic part. Ithas been shown previously that NTE catalytic activity wasnot regulated by cAMP based on the results of overexpressionof NTE in both prokaryotic and eukaryotic cells. The resultsof the in vitro test of binding of cAMP to the predicated simi-lar sequence of PKA regulatory subunits indicated that theN-terminal of NTE did not bind to the cAMP. It was sug-gested that the similar sequence in the region of N-terminalregulatory domain to PKA is only the similarity of structurein sequence but not of real equal functional significance(Dremier et al., 2003). However, it was not excluded thatthe lowering of intracellular cAMP may affect NTE activityand there existed the potential interaction of NTE andcAMP. The experiment of yeast two-hybridization (Y2H)found that the guanine nucleotide binding protein beta poly-peptide 2 (GNB2) and GNB2-like I protein, which werescreened from the human fetal brain library, can stronglyinteract with NTE. Co-immunoprecipitation analysis con-firmed the interaction of Gb2 protein and NTE in mammaliancells. The G protein signal pathway influencing factor, pertus-sis toxin, treatment in vivo, and the depletion of Gb2 by RNAinterference downregulated the activity of NTE but not its

9.2 MOLECULAR BIOLOGY OF NEUROPATHY TARGET ESTERASE 113

expression in the levels of protein and mRNA (Chen et al.,2007a). These results indicated that both the treatment of per-tussis toxin and the inhibition of Gb2 expression can affectthe cAMP levels, which further regulate the NTE catalyticactivity. The overexpression of peptides of NTE containingthe binding region of cAMP in the cells causes accumulationof recombined polypeptides, suggesting the cAMP may affectthe normal configuration of NTE in the cells (Glynn, 2005).Therefore, the regulation of NTE by cAMP and the inter-action of NTE and cAMP need to be further investigated.

Also using the Y2H system to screen the proteins that reactwith NTE, we found that androgen receptor-associatedprotein 54 (ARA54) can react strongly with the NTE regulat-ory domain (Chen et al., 2005). It is known that ARA54 isa ubiquitin protein ligase of the ubiquitin-protease pathway,regulating the protein levels and inducing the ubiquitinationof ARA54 itself (Ito et al., 2001). Both ARA54 and theN-terminal of NTE contain the destruction-box (D-Box),which is the ubiquitin recognition signal, suggesting thatNTE expression may be regulated by the ubiquitin-proteasepathway. Our recent studies revealed that the ubiquitin-protease pathway was involved in the degradation of NTEand there is an interaction of NTE with ARA54. Over-expression of ARA54 promoted the degradation of NTE,while the depletion of ARA54 by RNA interference pre-vented NTE degradation.

NTE was probably regulated by other proteins from theviewpoint of the metabolism. Owing to the degradation ofphosphocholine catalyzed by NTE, the most possible NTEfunctional regulation is from the need for maintaining thebalance of synthesis and degradation of phosphocholine. Inyeast, when the temperature is increased to 378C from308C, the synthesis of phosphocholine increases and theintracellular GPC levels increases correspondingly to main-tain the homeostasis of phosphocholine through the role ofNTE as a phospholipase, which makes yeast overexpressingNTE able to live at a higher temperature (Murray andMcMaster, 2005). Recent studies found that the phospholipidbinding protein Sec14p in cytoplasm in the yeast not onlybinds to phosphocholine to inhibit the synthesis of CDP-choline but also functionally promotes the catalytic reactionof NTE to accelerate the degradation of phosphocholine,which plays a dual regulatory role (Murray and McMaster,2005). When CTP-phosphocholine cytidylyltransferase(CCT) was overexpressed, the synthesis rate of phosphocho-line increased but its levels remained unchanged and the GPClevels increased, which was regulated by the enzymes includ-ing iPLA2 and NTE. Overexpression of NTE caused increaseof GPC, which can be blocked through the inhibition of NTEoverexpression by the reduction of choline absorption andCDP-choline formation. There also was antagonistic actionbetween NTE and CCT to maintain the homeostasis of phos-phocholine (Jackowski and Fagone, 2005). In addition, acti-vation of protein kinase C (PKC) inhibits mRNA expression

of NTE and decreases the enzyme activity, which may berelated to PKC activated phospholipase D, and then speedsup the degradation of phospholipid to phosphatidic acidsand choline to maintain the homeostasis of phosphocholine.The degradation of phosphocholine to glycerphosphocholineand fatty acids was reduced by inhibition of the NTE catalyticactivity (Gallazzini et al., 2006).

9.3 MOLECULAR TOXICOLOGY OFNEUROPATHY TARGET ESTERASE

9.3.1 NTE and OPIDN

NTE was discovered while the mechanism of OPIDN wasbeing studied but it was found that all OPs did not inducedelayed neurotoxicity. Martin K. Johnson noticed that phos-phonate esters, sulfonate esters, and carbamates can inhibitNTE activity but cannot induce delayed neurotoxicity. Hespeculated that the neuropathic OPs induced the delayedneurotoxicity only after the phosphorylated NTE was aged,which makes the enzyme catalytic activity unable to be reac-tivated (Johnson, 1974).

9.3.2 The Pathway of NTE Aging

The steps of NTE aging include detachment of the side groupR (usually an alkyl group) from the active site of the phos-phorylated NTE molecule, and a substituent group with anegative charge then binds to the site, which makes the phos-porylated NTE negatively charged; meanwhile, the detachedside group binds to the amino acids (site Z) near the active site(Fig. 9.3, Path 2; Johnson, 1974). The “aging” mentionedhere means the change of the enzyme activity, which is con-sistent with the concept of the aging in enzymology andmeans that the inhibition of enzyme activity cannot bereversed. The inhibited NTE catalytic activity by neuropathicOPs cannot be restored by even KF, a nucleophilic reactiva-tor, which is usually used to restore the phosphorylated butnot aged enzyme activity. The condition of OPIDN is thatNTE was inhibited (inhibition rate .70%) and then agedone or two days after dosing. If NTE was only inhibited bythe OP but not undergoing an aging reaction, no OPIDNoccurred. For example, an OP like phenyl dipentyl phosphi-nate, which inhibits NTE activity only but does not age thephosphorylated enzyme, cannot induce OPIDN. Johnsonspeculated that the aged NTE probably changed either theprotein feature or the reaction environment to cause OPIDN(Johnson, 1993).

The mechanism of NTE inhibition by OPs is to be under-stood gradually along with the study on the NTE active sites.NTE cloning provides the studies of molecular mechanismsof NTE aging with new clues. The studies showed that thereaction of OP with NEST expressed in prokaryotic cells

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was very similar to that of OP with NTE in the tissues. Thetest of the binding of [3H] labeled diisopropyl fluorophos-phate (DFP) to NEST as well as the determination ofenzyme-hydrolysis isotope revealed that the reaction site ofNEST aged by DFP was at Ala955, and Asp1044, which isprobably the binding site of the side group; however, therealso exist other sites, among which Asp1004 is a possiblesite (Atkins and Glynn, 2000). The mass chromatographyconfirmed that the isopropyl group, a side group of DFP,really transferred within molecules when DFP binding toSer966 after NEST inhibition (Kropp et al., 2004).

The hypothesis of NTE aging proposed by Johnson wasextensively recognized, but whether there are any differentmechanisms of NTE aging by different OPs remainsunknown. The enzyme dynamic analysis of NTE inhibitionand aging by mipafox revealed that KF can restore the activityof NTE aged by mipafox only in acidic conditions (pH ¼5.2) (Milatovic and Johnson, 1993); this reactivation doesnot happen in alkaline conditions (pH ¼ 8.0; Richardson,1995). Therefore, the process of inhibition and aging ofNTE by mipafox is reversible, which cannot be explainedby the simple mechanism of the side group intermoleculartransfer. It was found recently that no aging with side groupintermolecular transfer interactions happened after mipafoxinhibited NEST expressed in prokaryotic cells; however, thedeprotonization happened, which makes the phosphorylatedenzyme with a negative charge be aged (Fig. 9.3, Path 1;Kropp et al., 2004). This deprotonization process is reversibleand regulated by pH values; no aging occurs in acidic con-ditions (e.g., pH ¼ 5.2) while aging occurs completely inalkaline conditions (e.g., pH ¼ 8.0; Kropp et al., 2004).The mechanisms of NTE aging may be different with differ-ent OPs, which include at least two pathways; one is the

classical side group transfer and the other is the newly discov-ered deprotonization.

9.3.3 Mechanisms of OPIDN Related to NTE

The inhibition and then aging of NTE were considered theessential condition for OPIDN; some studies showed thatno OPIDN signs were observed in hens dosed for twoweeks to prolong the inhibition of NTE with OP that hasno capability of aging. It was speculated that NTE plays animportant role in development of the nerve system, but itsesterase activity seems to be redundant for adult vertebrates.The decisive condition for OPIDN is that neuropathic OPsmodify the active site of NTE by a negative charge group,which means (1) OP makes the loss of non-esterase activityfunction that is essential for neurons or axons although themechanism remains unclear up till now; and (2) NTE ismaybe not necessary for adult animals. However, OPmakes a negative charge group modify the active site andNTE gained a toxic function, causing OPIDN (Glynn, 2000).

Heterozygote mice with NTE gene knockout displayhyperactivity signs instead of OPIDN. The gene knockoutmice die easily after challenged by ethyl octylphosphono-fluoridate (EOPF), an NTE catalytic activity inhibitor. Onlythe enzyme activity inhibition but not aging is required forthis lethality (Winrow et al., 2003). It was therefore concludedthat NTE aging is unnecessary for the NTE toxicity to miceand the toxicity is not a gain-of-function (Winrow et al.,2003). However, Glynn thought that the gain-of-function ofNTE could not be excluded because the signs of the delayedneurotoxicity are different in mouse and chicken, and themouse is not the typical animal model for OPIDN. Basedon the different toxic signs observed in mouse, chicken,

Figure 9.3 Pathway and process of aging of neuropathy target esterase. Adapted from Kropp, T. J. et al. 2004. Biochemistry43(12):3716–3722. Modified with permission.

9.3 MOLECULAR TOXICOLOGY OF NEUROPATHY TARGET ESTERASE 115

and fruit fly with SWS mutation, Glynn presumed that NTEas the target in different organisms causes different toxicsigns through different mechanisms (Glynn, 2003).

Although it was found that the hydroxyl group of Ser966 atthe active center of NTE was phosphorylated and the sidegroup of OP linked to the inhibited NTE was detached orthe OP group underwent deprotonization, it left a negativecharge substitute group attached to the active site (aging reac-tion) to block the reactivation of the enzyme activity. This isthe prerequisite for OPIDN, but the inhibition and aging ofNTE were the early events (one to two days after dosing)and then the inhibited enzyme activity was gradually restored;however, the biochemical events that occur during the pro-cess before the signs of OPIDN are observed two to threeweeks after exposure remain unidentified.

There seems to be enough facts to establish a relationshipbetween NTE and OPIDN based on the role of NTE in themetabolism of membrane lipids and membrane structure. Inneurons, NTE was inhibited by OPs, which produce ERstress and disturb the homeostasis of ER membrane lipids,while almost all substance in the neuron axons was trans-ported rapidly through ER, which was inevitably disarrangedduring OPIDN. For large animals such as humans, cats,cattle, and chickens, the substances produced in the body ofthe neurons took several days to reach the terminal of thelong axons, which may be the reason the long axons wereinjured most easily, while the obvious characteristics of histo-pathology of OPIDN is the degeneration of the peripheralnerve long axons (Glynn, 2006, 2007). In addition, inhibitionof NTE activity in glia cells also disturbed the metabolism ofmembrane lipids and affected the interaction of neurons andglia; the terminal of long axons become the most easilyinjured part (Glynn, 2006, 2007). Therefore, it was specu-lated that the inhibition of NTE by OP caused a disturbanceof lipid homeostasis and ER function in nerves, includingneurons and glia, to hinder the substance transportation ofaxons and the interaction of neurons and glia cells and finallycaused the degeneration of distal axons (Fig. 9.4). In addition,based on the facts that NTE possess lysophospholipaseactivity and affect the lipid metabolism in tissues, anotherhypothesis was suggested that OPIDN was induced by thechange of phospholipid metabolism and its signal transduc-tion pathway in some specific parts by the OPs (Quistadand Casida, 2004). However, a recent study showed that thehomeostasis of phosphatidylcholine and lysophosphatidyl-choline was not disrupted during TOCP-induced delayedneurotoxicity in hens although the inhibition of NTE, lyso-phospholipase, and phospholipase B activities and decreaseof GPC levels were observed (Hou et al., 2008).

The role of NTE in OPIDN has been confirmed by mol-ecular genetics. No neurodegenerative signs were observedin the NTE2/þ knockout mice while the NTE activity wasonly 50% of that in normal mice (Moser et al., 2004;Winrow et al., 2003). However, there is only 10% of normal

NTE activity in the mice with brain-specific knockout of theNTE gene (Akassoglou et al., 2004) and the mice displayedneurodegenerative signs, which indicated that only whenmore than a certain amount of NTE was inhibited can the neu-rodegeneration be induced. Axonal degeneration and hind-limb paralysis were observed in the brain-specific NTEknockout mice at six to nine months, which is similar tothat of OPIDN. It indicated that the inhibition of NTE mustreach a higher level for the OPIDN (more than 70% ofNTE was inhibited in the hen model). It was found thatNTE mutation lead to motor neuron disease (MND; Rainieret al., 2008). The gene types of the patients from the membersof the two families of the MND were analyzed and found thataffected subjects in the consanguineous kindred were homo-zygous for disease-specific NTE mutation c. 3034A to G thatdisrupted an interspecies conserved residue (M1012V) in theNTE catalytic domain. Two places of mutations in the cDNAcoding sequences of NTE in the affected subjects from thenonconsanguineous family were found to be compound het-erozygotes: one allele had a c. 2669G to A mutation, whichmakes Arg890 become His890; and the other allele had aninsertion (c. 2946_2947insCAGC) causing frameshift andprotein truncation (p. S982fs1019). NTE mutations werefound in the unrelated MND patients and the mutationswere located in the encoding region of the catalytic domainof NTE. Therefore, this MND was NTE specific and calledNTE-related MND (Rainier et al., 2008).

9.3.4 Application of NTE in the Prediction of Toxicity

The in vitro test system was used to determine the inhibitionof NTE by OPs, which became a fast and economic methodfor prediction of OPIDN and distinguishing delayed neuro-toxicity and acute toxicity since the hypothesis that NTEinhibition and then aging is the key step for the initiationof OPIDN was proposed.

Figure 9.4 Hypothesis of the mechanism of OPIDN resultingfrom the NTE inhibition by neuropathic organophosphorus com-pounds. From Glynn, P., 2007. Arh Hig Rada Toksikol58(3):355–358. With permission.

116 MOLECULAR TOXICOLOGY OF NEUROPATHY TARGET ESTERASE

It is a prerequisite to do this work to select cultured cellsthat possess NTE activity. Nostrandt et al. (1992) selectedhuman neuroblastoma SK-N-SH and its subclone cell lineSH-SY5Y, and PC12 cell to do the test and found that NTEactivity is higher in SH-SY5Y cells. The inhibition andaging of NTE in SH-SY5Y cells by mipafox were similarto that in the hen brain homogenate, while non-neuropathicparaoxon did not inhibit NTE in the cells (Nostrandt andEhrich, 1992). The above results suggest that the SH-SY5Ycell is perhaps an ideal model for in vitro testing to distinguishneuropathic OPs from non-neuropathic OPs. Other exper-iments also confirmed that the inhibition of NTE in the differ-entiated SH-SY5Y cells by mipafox, aldicarb, and verapamilwas similar to that in hen brain homogenates, which indicatedthat SH-SY5Y can be used to test the inhibition of NTE andthe result is consistent with that observed in the hen brainhomogenate (Nostrandt and Ehrich, 1993). More results ofin vitro tests suggested that the SH-SY5Y cell is a potentialmodel for prediction of in vivo neurotoxicity (Ehrich, 1995;Ehrich et al., 1994). Meanwhile, the OPs that caused acutetoxicity by inhibiting AChE in the animal model can inhibitAChE activity in SH-SY5Y cells (Ehrich, 1995). By com-parison, it was found that the SH-SY5Y cell was a useful invitro model to effectively distinguish esterase-inhibiting OPneurotoxicants (Ehrich et al., 1997). The above resultssuggest that the cultured cells not only predict the potentialability of the OPs to induce delayed neurotoxicity, but alsorecognize the OPs that produce acute toxicity and those pro-ducing delayed neuropathy.

Different animals show different rates of inhibition ofNTE by a given OP; thus, the sensitivity of NTE in differentanimals to the OP is different. Similarly, cultured cells fromdifferent tissues have different sensitivity to cytotoxicity ofOPs. By comparison of human neuroblastoma SH-SY5Ycells and mouse neuroblastoma NB41A3, it was found thatNB41A3 is more sensitive to the same concentration ofOPs than the SH-SY5Y, which may be relative to the differ-ence of the metabolism and the activity of enzyme in the twotypes of cells (Ehrich and Veronesi, 1995). The activity ofNTE in NB41A3 is lower than that in SH-SY5Y. The factthat NTE activity is lower in NB41A3 is consistent withthat observed in mice (Ehrich, 1995; Veronesi et al., 1997),which is the theoretical base for in vitro prediction of thein vivo toxicity difference of OPs.

Although the above results indicate that there is greatpotential for cultured cells to be used to predict the toxicityof chemicals, the OPs tested did not include their precursors.It was known that NB41A3, which has a high metabolicactivity, is more sensitive than SH-SY5Y to the precursorsof chemicals (Veronesi and Ehrich, 1993). It should considerthat how to test the toxicity of precursors of the chemicalsin vitro using cell models to consistent with the results ofin vivo tests. Barber et al. (1999a) reported that NTEinhibition was enhanced by adding active substance (e.g.,

bromide) into the cultured cells to activate the precursors ofthe chemical metabolized, which is getting close to the realcircumstance in vivo study, and using rat liver microsomescan reflect more the real circumstance than the bromideaddition (Barber et al., 1999b). More experiments of acti-vation of chemical precursors were carried out and comparedwith the results of experiments of brain homogenates of hens,which confirmed the above results. More obvious inhibitionof AChE in cells was found after the activation of activatorsto the precursors (Barber et al., 1999b). The above resultslay a solid theoretical and practical foundation of using cellmodels to predict the toxicity of chemical precursors.

9.4 PROSPECTS OF MOLECULARTOXICOLOGY OF NTE

All facts, from the discovery of NTE to the proposition ofhypotheses for mechanisms of OPIDN, were closely relatedto the understanding of molecular features of NTE. The clon-ing, catalytic characteristics, cell location, and physiologicalfunction of NTE are the prerequisite for understanding themechanisms of OPIDN, which is also helpful to understandthe molecular toxicology of NTE. Therefore, to understandthe NTE at the molecular level is an important step toreveal the essence of NTE molecular toxicology.

There is little systematic research on NTE expression andregulation from the viewpoint of NTE molecular biology. Wehave found that the ubiquitin-protease pathway and the lyso-some pathway were involved in the degradation of NTE at theprotein level (Long et al., 2009), but little was known aboutthe degradation and regulation of NTE at RNA levels. Itwas found that PKC activation decreased NTE mRNAlevels (Chen et al., 2007b), but it was unclear how to regulatethe transcription of NTE by PKC. The promoter of NTEneeds to be cloned to understand the transcription and regu-lation of NTE and analyze the effects of factors that regulateNTE transcription and their interaction in order to establish afoundation for understanding the regulation of NTE activity.Besides, the total protein amount of NTE can be regulated,which includes regulation in the mRNA and protein levels.It is also very important for the network regulation by inter-action of NTE and other molecules. Whether cAMP orcGMP can bind to the three cAMP binding domains in theN-terminal of NTE to regulate the activity of the enzymeneeds to be studied. Whether there exists the fact that post-translational modification (e.g., covalent modification suchas phosphorylation) regulates the NTE activity still remainsunknown. This all needs to be further investigated.

In NTE molecular toxicology, most studies focused on themechanisms of OPIDN, which is closely related to NTE. Thefuture studies about the mechanisms of OPIDN must bedeveloped deeply in the field of NTE molecule. At leasttwo important questions need to be answered. One is about

9.4 PROSPECTS OF MOLECULAR TOXICOLOGY OF NTE 117

the molecular mechanism of NTE aging; why only organo-phosphorus esters induce delayed neurotoxicity while phos-phate, sulfonic esters, and carbamates cannot? The other isabout the mechanism of differences in sensitivity toOPIDN in different species; why do susceptible animalssuch as humans, cats, and hens display the OPIDN signsafter being exposed to neuropathic OP while rodents suchas mice, rats, and rabbits do not? Is OPIDN related to thedifference of the activity, function, and sensitivity of NTEor the body anatomy structure in species, or both? In addition,high specific NTE activity was found in the nerve system,and higher activity of NTE was also found in non-neuronaltissues such as lymphocyte, kidney, and intestine. The phys-iological role of the NTE in these non-neuronal tissues andthe related toxicology have yet to be explored.

9.5 CONCLUSIONS

Although the relationship of NTE and OPIDN has been estab-lished and NTE is considered to be the primary target ofneuropathic OPs and the inhibition and aging of NTE is theinitiation event of OPIDN, the exact events that occurduring OPIDN, especially what happens during the timeafter NTE inhibition and before the clinical signs appearremains unknown. NTE was found to be a lysophospholipaseand to hydrolyze phosphatidylcholine and lysophosphatidyl-choline in mice; however, no disruption of the homeostasisof phosphatidylcholine and lysophosphatidylcholine wasobserved in hens with delayed neurotoxicity induced byTOCP. Therefore, whether the change of phospholipidmetabolism was involved in the mechanism of OPIDN stillremains unclear.

There are one transmembrane and three cAMP-bindingdomains in the N-terminal of NTE molecule. However,until now, cAMP binding to these domains or the regulationof NTE activity by cAMP has not been observed in prokary-otic cells and eukaryotic cells. The functional roles of thedomains are still to be unveiled.

ACKNOWLEDGMENT

This work was supported by a grant from the National NatureScience Foundation of China (30870537).

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