Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Acetylcholine is the primary neurotransmitter of the cholinergic system, and its activity is regulated through hydrolysis in nerve synapses by acetylcholinesterase (AChE). The termination of nerve impulse transmission is accomplished through the degradation of acetylcholine into choline and acetic acid by AChE. Although the importance of AChE in the function of the nervous system has been recognized for more than 80 years, its role in development remains enigmatic (Robertson, 1987; Layer, 1996; Andres et al., 1997; Bigbee et al., 1999; Brimijoin and Koenigsberger, 1999; Lassiter et al., 1998). AChE activity is found in brain regions that are devoid of cholinergic neurons, acetylcholine, and acetylcholine receptors (Greenfield, 1984). AChE is transiently expressed during discrete periods of neural development of the thalamocortical pathways, and transient AChE activity correlates with the specific growth of thalamic axons into the cortex and synaptogenesis with cortical neurons (Robertson and Yu, 1993). In addition, significant sequence similarity exists between AChE and cell adhesion proteins that function in morphogenic phenomena. These observations have led to the hypothesis that AChE may play key roles in neural development. Robertson and Yu (1993) proposed that AChE may be bound to a proteolytic enzyme that aids the growing axon in maneuvering through cortical neuropil to reach its target.

The many important functions attributed to the cholinergic system suggest that sustained life is not possible in the absence of AChE. Deletion of AChE activity in Drosophila through mutagenesis resulted in embryonic lethality (Greenspan et al., 1980). Chemicals with anticholinesterase activity such as organophosphorus and carbamate pesticides are lethal to humans and animals after acute exposure (Doctor et al., 1991; Taylor and Radic, 1994; Cowan et al., 1996; Massoulié et al., 1996; Taylor, 1996; Mileson et al., 1998). Inhalation of the chemical warfare agent Sarin by 5000 people in the Tokyo subway resulted in acute respiratory failure and the death of 12 (Nagao et al., 1997). The inhibition of AChE by organophosphorus poisons produces a massive outpouring of secretions, neuromuscular block, and central depression of respiration (Namba et al., 1971), yet chronic exposure to organophosphates, such as metrifonate for the treatment of Alzheimer's disease, results in accommodation and is not lethal (Cutler et al., 1998).

Mice lacking expression of AChE were generated to investigate the contribution of AChE to development and to explore the potential compensatory role of butyrylcholinesterase (BChE; EC 3.1.1.8). AChE and BChE arise from distinct genes and have about 50% sequence identity. Both enzymes hydrolyze acetylcholine. Contrary to expectation, mice without AChE activity survived to birth and for up to 3 weeks after birth. Therefore, the question was asked whether mice without AChE are sensitive to the organophosphate diisopropylfluorophosphate (DFP).

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Generation of AChE / Mice. A lambda FIX II clone containing the complete mouse ACHE gene was isolated from a mouse strain 129SVJ genomic library (Stratagene, La Jolla, CA). The targeting vector (Fig. 1) for disrupting the ACHE gene was introduced into R1 embryonic stem cells by standard methods (Wurst and Joyner, 1993; Wilder et al., 1997). Of 200 embryonic stem cell colonies screened by Southern blotting, four contained the desired disrupted allele. Genomic DNA digested with XbaI, NheI, XhoI, or BamHI and hybridized with the probe indicated in Fig. 1 or with a probe for the neo gene confirmed that the null allele was present in these four colonies and that additional copies of the targeting vector were not randomly integrated into their genomes (data not shown). Eleven chimeric mice were generated, and two of these transmitted the ACHE mutant allele in their germline (Xie et al., 1999). The phenotype of the two lines was indistinguishable. Chimeric mice were mated to strain 129sv mice (Taconic 129S6/SvEvTac) to maintain the ACHE knockout animal in a 129sv background. Mice were fed Teklad LM-485 mouse/rat irradiated diet (catalog number 7912) containing 5% fat and 19% protein (Harlan, Madison, WI). Experimental protocols adhered to the guidelines of the U.S. National Institutes of Health for the care and use of laboratory animals.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Targeting strategy for deleting the ACHE gene. Top line, structure of the mouse ACHE gene (Li et al., 1991). Black boxes represent exons 1 to 6. The hatched region between exons 5 and 6 is a retained intron that can be transcribed into mRNA. Exons 5 and 6 are alternatively spliced. The most abundant AChE originates from splicing to exon 6. Middle line, targeting replacement vector. The 5.2- and 0.9-kb segments of the ACHE gene were inserted into the plasmid MJK-KO on either side of the neomycin phosphotransferase gene cassette (neo). The plasmid also contains an HSV thymidine kinase gene cassette (tk) for negative selection. Bottom line, structure of the ACHE knockout allele from which 5 kb of the ACHE gene has been deleted, including exons 2 to 5. The probe used to identify targeted cells by Southern blotting is shown. The 6.5- and 5.0-kb BamHI fragments recognized by the probe in the wild-type and targeted alleles are indicated.

Measurement of AChE and BChE Activity. Tissues from 10- to 12 day-old mice were extracted with 50 mM potassium phosphate, pH 7.4, containing 0.5% Tween 20 and assayed for activity with 1 mM acetylthiocholine. Tissues were extracted with buffer containing 0.5% Tween 20 rather than the commonly used Triton X-100 (Massoulié and Toutant, 1988; Feng et al., 1999) because Triton X-100 inhibits 90 to 95% of mouse BChE activity. Heart and skeletal muscle (quadriceps) pellets from the first extraction were re-extracted with buffer containing 1 M NaCl to extract the collagen-tailed forms of AChE and BChE. The activity remaining after AChE was inhibited with 0.01 mM 1,5-bis(4-allyldimethylammonium phenyl)-pentan-3-one was BChE. AChE activity in intestine was calculated after inhibition of BChE with 0.1 mM tetraisopropylpyrophosphoramide (iso-OMPA).

Gel Electrophoresis. Nondenaturing gradient gels of 4 to 30% polyacrylamide and 0.75-mm thick were made in a Hoefer apparatus. Then, 3 µl of serum was loaded per lane from two wild-type, two AChE +/, and three AChE / mice. The control samples, human serum and fetal bovine serum, also were loaded at 3 µl/lane. The upper buffer contained 600 ml of 0.021 M Trizma base, 0.023 M glycine with unadjusted pH 9.0; the lower buffer contained 4.5 L of 0.06 M Tris-HCl, pH 8.1. Electrophoresis was at 4°C for 40 h at a constant voltage of 100 V. The staining buffer (Karnovsky and Roots, 1964) produced a brown precipitate in locations of cholinesterase activity. The staining buffer contained 1.7 mM acetylthiocholine iodide when it was desired that all cholinesterase bands be seen but contained 2 mM butyrylthiocholine iodide when only BChE activity was visualized. To specifically visualize bands of AChE activity, the gel was incubated for 30 min with 0.01 mM iso-OMPA, an inhibitor of BChE, before the addition of acetylthiocholine iodide.

Toxicity Studies. The 12-day-old mice were injected i.p. with diisopropylfluorophosphate (DFP) dissolved in PBS. The DFP solution was prepared just before use because this organophosphate is unstable in aqueous buffer. The DFP dose of 2.5 mg/kg was chosen after it was determined that 12-day-old wild-type mice survived this dose but that a slightly higher dose was lethal to a few wild-type animals. Bambuterol (Astra Draco AB, Lund, Sweden), a carbamate prodrug of the antiasthma drug terbutaline (Tunek and Svensson, 1988), was dissolved in PBS and injected i.p. Bambuterol is a specific inhibitor of BChE.

Histology. The 12-day-old animals were perfused with 4% buffered formalin before tissues were removed. Brains were stored in 10% buffered formalin for 24 h at 4°C and then in 0.1 M PBS containing 20% sucrose for 2 to 3 days. To facilitate sectioning, each specimen was embedded in Tissue-Tek OCT Compound (Sakura Finetek USA, Inc., Torrance, CA) and flash frozen in 2-methylbutane submerged in a dry ice/ethanol bath. The blocks were stored at 80°C until sectioning. Frozen brains were cut on a cryostat into 20-µm sections. Sections were mounted onto Fisherfinest Premium Superfrost microscope slides (Fisher Scientific), which are electrically charged. Slides were air-dried and then stored at 80°C until they were stained for AChE activity by the method of Karnovsky and Roots (1964) and counterstained with hematoxylin. The details of the AChE staining procedure are as follows. The staining buffer was freshly prepared just before use by mixing 30 ml of 0.2 M maleate (pH adjusted to 6.0 with 1 M NaOH), 2.5 ml of 0.1 M sodium citrate, 5.0 ml of 0.030 M cupric sulfate, 5.0 ml of 0.005 M potassium ferricyanide, and water to a total of 50 ml. The staining buffer was filtered through a 0.22-µm filter to remove particulates. When the buffer was not filtered, the slides had dark spots. Four slides were incubated in 29 ml of the filtered staining buffer containing 0.1 mM iso-OMPA (0.145 ml of 20 mM iso-OMPA in water). The purpose of incubating in iso-OMPA was to specifically inhibit BChE. After 20 min of incubation with gentle shaking, 0.29 ml of 0.2 M acetylthiocholine was added. The incubation with 2 mM acetylthiocholine was for 2.5 h at 25°C. At the end of the incubation period, the slides were washed with water 10 times and then fixed in 4% formalin in PBS for 15 min at room temperature. The slides were dipped in hematoxylin for 40 s and then five times each in 80, 95, 100% ethanol, and xylene and covered with a drop of glue and a coverslip.

Electron Microscopy. The 12-day-old mice were perfused trans-cardially with physiological saline containing 2.5% glutaraldehyde and 2% paraformaldehyde in phosphate buffer. The quadriceps muscle was dissected and fixed overnight in the same fixative. The midportion of the muscle was isolated, postfixed in buffered 1% osmium tetroxide for 1 h, dehydrated, and embedded in Araldite. Thin sections were stained with uranyl acetate and lead citrate and viewed under the electron microscope.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

A strategy for disrupting expression of AChE using homologous recombination was achieved through the deletion of 5 kb of the mouse ACHE gene (Fig. 1). Deletion of exons 2 through 5 removed 93% of the 583 amino acids comprising AChE. It was expected that ACHE mutant animals would have gross abnormalities or show a maturation arrest in utero at a time when AChE activity was essential for continued development. Although ACHE mutant Drosophila were not viable, the possibility existed that nullizygous ACHE mice would be viable because BChE is not present in Drosophila.

To demonstrate that the recombination event had generated the anticipated ACHE null allele, genomic DNA from 12-day-old mice was analyzed by Southern blotting (Fig. 2A) and polymerase chain reaction (Fig. 2B). The results supported the conclusion that the tested 12-day-old mice had no wild-type ACHE allele but had two null ACHE alleles.

View larger version (72K): [in this window] [in a new window]   Fig. 2.   Evidence that the ACHE gene has been deleted. A, Southern blot of genomic DNA isolated from 12-day-old AChE +/+, AChE +/, and AChE / mice. DNA was digested with BamHI and hybridized with a probe located outside the targeted region. B, polymerase chain reaction analysis of mouse DNA. Primers were in intron 1 and exon 2 for the wild-type allele and in exon 1 and neo for the knockout allele.

AChE enzyme activity was analyzed in serum from blood of wild-type, heterozygous, and nullizygous animals using nondenaturing polyacrylamide gels stained for total cholinesterase activity (Fig. 3A) and specifically for AChE activity (Fig. 3B). Control 12-day-old wild-type mice demonstrated at least nine bands of cholinesterase activity: six bands were identified as BChE and three were identified as AChE by selective inhibition of BChE with 0.01 mM iso-OMPA (Fig. 3B), as well as by selective inhibition of AChE with 0.03 mM 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one (not shown) and by selective staining for BChE activity with butyrylthiocholine (not shown). The bands of AChE activity correspond with monomeric, dimeric, and tetrameric forms. The three AChE bands were less intense in AChE +/ mice and were missing in sera from nullizygous AChE mice.

View larger version (88K): [in this window] [in a new window]   Fig. 3.   Evidence for the absence of AChE activity. A, nondenaturing polyacrylamide gel of blood serum samples. The gel was stained with acetylthiocholine (Karnovsky and Roots, 1964) to reveal total AChE and BChE activity. Human (Hu) serum contains only BChE. FBS contains only AChE. Wild-type mouse serum contains 70% BChE and 30% AChE. The top double arrow points at tetramers of AChE, the middle double arrow points at a faint band of dimers of AChE, and the brackets indicate a broad band of monomeric AChE. These bands are easier to see in the gel below. B, nondenaturing polyacrylamide gel stained for AChE activity. The gel was incubated in 0.01 mM iso-OMPA, an inhibitor of BChE, for 30 min before it was treated with 1.7 mM acetylthiocholine to reveal AChE activity. A residual band of tetrameric BChE in lane Hu is due to incomplete inhibition of the most intense band of BChE. The top double arrow points at tetrameric AChE, the middle double arrow points at dimeric AChE, and the brackets indicate monomeric AChE in wild-type and AChE +/ mouse sera. These bands of AChE activity are absent in AChE / mouse sera.

Tissue levels of AChE were evaluated using a spectrophotometric assay with acetylthiocholine and a specific AChE inhibitor to estimate the proportion of AChE and BChE activity (Table 1). Cholinergic neurons innervate the brain, heart, eye, glands, trachea, spleen, stomach, small bowel, colon, kidney, urinary bladder, external genitalia, and skeletal muscle (Lefkowitz et al., 1996), and these tissues all contain AChE. In wild-type mice, the highest AChE activity was in brain, followed by serum and intestine. AChE +/ mice had less AChE activity than wild-type mice in all tissues tested. AChE / mice had no detectable AChE activity in brain, serum, intestine, heart, lung, muscle, and liver. AChE expression in the nullizygote was further explored by staining sections of mouse brains for AChE activity. Wild-type and AChE +/ mouse brains showed intense AChE activity in the caudate and putamen, whereas AChE / brain had no AChE activity (Fig. 4).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Brain sections stained for AChE activity. A, wild-type +/+. B, heterozygous +/. C, nullizygous /. Magnification, 12.5×.

Phenotype. Observations of movement, behavior, and morphologic features were initiated to identify possible focal defects in activity or function. The AChE / mice were indistinguishable from their littermates at birth through visual inspection. However, weight gain and growth were retarded, and at 7 days, a clear difference was noticeable between nullizygous animals and normal littermates (Fig. 5B). Newborn litters were weighed daily beginning on day 2 after birth, and results were charted. The weights of AChE / mice lagged behind those of wild-type littermates at all time points and began to fall 1 to 2 days before death. Nullizygous animals were observed to nurse, and their stomachs contained milk curds at the time of death. However, they appeared emaciated and dehydrated.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Life span of AChE / mice and weight gain relative to littermates. A, Kaplan-Meier survival curve of 63 AChE / mice. Of the AChE / mice, 16% were born dead compared with 8% of AChE +/ and 5% of wild-type mice. B, average postnatal weight of 18 AChE / mice compared with 57 AChE +/+ and +/ mice. Each point is the average value for 18 or 57 animals. The data for wild-type and +/ mice are pooled because these animals are indistinguishable by weight. All AChE / mice in this protocol died by day 14.

View larger version (121K): [in this window] [in a new window]   Fig. 6.   Size comparison of a 12-day-old AChE / mouse (foreground) and a 12-day-old wild-type littermate. A video depicting the characteristic tremor can be viewed at http://www.unmc.edu/Eppley/f_lockr.html using the free software Quicktime or RealPlayer.

Histology. The heart, lungs, thymus, liver, spleen, adrenal glands, kidneys, pancreas, small and large intestines, stomach, and reproductive organs from AChE / animals were examined and showed no external abnormalities. The cerebral hemispheres of the brain were translucent in 12-day-old AChE / mice but were opaque in normal littermates. The cerebellum and brain stem showed no external abnormalities. Light microscopic examination of the small and large intestines from AChE / animals showed no fibrosis or hypertrophy of the muscularis. Intermyenteric nerves were present, although no AChE activity was apparent by staining. The normal architecture of the adrenal was maintained. The lungs showed normal expansion without inflammatory infiltrate. The liver architecture was intact. Normal striations were apparent in the heart myocytes. No abnormalities were present in the stomach, spleen, pancreas, kidneys, thymus, or bladder. No histologic abnormalities were seen in any of the organs from heterozygous animals.

Neuromuscular Junction. Electron microscopy showed that the neuromuscular junction of AChE nullizygous animals was well formed (Fig. 7) and normal in size. Excessive encasement of the normal sized nerve termini by Schwann cell processes was not seen. The nerve termini contained synaptic vesicles and mitochondria. A few termini contained membranous material. There was no evidence of degeneration of the junctional folds.

View larger version (167K): [in this window] [in a new window]   Fig. 7.   Electron micrograph of the neuromuscular junction of the quadriceps muscle of a 12-day-old AChE / mouse. The nerve terminal (N) contains synaptic vesicles and mitochondria. Junctional folds in the postsynaptic muscle cell are well formed. A muscle cell nucleus lies below the junctional folds. Scale bar, 0.5 µm.

Survival without AChE. Mating studies of heterozygous animals were performed to determine whether nullizygous animals were born at the expected frequency of 25%. After 320 live births, it was determined that AChE / mice were born at a frequency of 20% (Table 2), suggesting that about one-fourth of the AChE / fetuses died in utero and were resorbed. The large proportion of live births indicated that AChE was not essential during embryogenesis and fetal development or that compensatory mechanisms existed. Two possibilities were considered, including that maternal AChE may have crossed the placenta and supported survival of the fetuses in utero and that BChE replaced AChE in developmental activities. No AChE activity was found in the sera of fetuses collected at day 16.5 post coitus, suggesting there was no maternal transfer of AChE.

Toxicity. It is generally agreed that the acute effects of poisoning by organophosphates are due to inhibition of AChE activity (Taylor, 1996; Pope, 1999). Because AChE / mice have no AChE enzyme activity, it was of interest to know whether nullizygotes would be resistant to the toxic effects of organophosphates. When the nonselective serine esterase and serine protease inhibitor, diisopropylfluorophosphate, was injected i.p. at a dose of 2.5 mg/kg, the nullizygous mice immediately collapsed and heartbeats ceased within 3 min. The wild-type 12-day-old littermates survived this dose (Table 3). Heterozygous animals had an intermediate sensitivity; half of the AChE +/ animals survived this dose. This result demonstrated that AChE is not the only target of organophosphorus poisons and that inhibition of other enzymes contributes to lethality. To test the possibility that BChE has a vital function in the nullizygote, a specific BChE inhibitor, bambuterol, was injected i.p. at a dose of 0.3 mg/kg. The nullizygote immediately showed signs of toxicity, was immobile by 6 min, and was dead by 10 min. Its normal littermates showed no signs of discomfort and survived. This result showed that in the absence of AChE, essential activities are provided by BChE or other serine esterases.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The finding that a mouse is able to live for up to 3 weeks postnatally despite being devoid of AChE activity in all tissues was totally unanticipated. This novel result is expected to influence viewpoints on the functional importance of AChE.

Development. The normal maturation and development of heterozygous animals demonstrated that the reduction in AChE activity to 50% of normal was sufficient to support development and growth. Although a complete absence of AChE did not impede embryonic organogenesis, it did restrict continued development after birth. Several possible explanations exist for this interesting combination of findings, including that the nullizygous animals are nutritionally deficient or are unable to obtain sufficient calories to sustain growth. The continuous tremulous motion of nullizygous animals may require a caloric intake beyond that of wild-type animals. Alternatively, AChE may contribute a structural or functional activity that is necessary to complete synaptogenesis and normal maturation. If the hydrolytic activity of AChE is necessary for embryogenesis and life-sustaining functions such as respiration, this function may be compensated by BChE or other serine esterases in the nullizygous animals.

Neuromuscular Junction. Less than 5% of AChE found in the body is anchored to the basal lamina of the neuromuscular junction through a collagen tail (Hall and Sanes, 1993; Feng et al., 1999). Humans with mutations in the collagen tail gene have end-plate AChE deficiency (Donger et al., 1998; Ohno et al., 1998). Their junctional folds degenerate, causing the loss of acetylcholine receptor. Mice lacking the gene for the collagen tail have no AChE or BChE in the neuromuscular junction, and their junctional folds also degenerate (Feng et al., 1999). The apparently intact neuromuscular junctions in AChE / mice suggest that the structural integrity of the neuromuscular junction might be maintained by the collagen tail alone or by collagen-tailed BChE.

Organophosphate Sensitivity. The finding that AChE / mice are supersensitive to the toxic effects of organophosphate shows that organophosphate inhibition of targets other than AChE leads to death. This result may not come as a surprise to toxicologists who have noticed that different organophosphorus pesticides cause different degrees of toxicity despite similar levels of AChE inhibition and have postulated the existence of toxicologically relevant sites of action in addition to AChE (Moser, 1995; Pope, 1999; Richards et al., 1999). What might these other targets be? The present work suggests that BChE might be a target in AChE / mice, but others are also likely. Members of the serine esterase family, including carboxylesterase, proline endopeptidase, leucine aminopeptidase, lipases, phospholipase, vitellogenin, Zn-dependent exopeptidase, cholesterol esterase, phosphatidylcholine-sterol acyltransferase, prolylcarboxypeptidase, and carboxypeptidase, are possibilities because they contain an active site serine (http://meleze.ensam.inra.fr/cholinesterase/). A serine esterase with no homology to the above proteins, neuropathy target esterase (Lush et al., 1998), is inhibited by certain organophosphates. Organophosphorus pesticides exert effects on proteins that have no active site serine: nicotinic receptor, muscarinic receptors, voltage-dependent chloride channel, -aminobutyric acid_A receptor, catecholaminergic pathways, and pathways that release neurotransmitters (Gant et al., 1987; Dam et al., 1999; Pope, 1999). The AChE / mouse should be useful for identifying toxicologically relevant targets.