Ellen G. Duysen

Rescue of the acetylcholinesterase knockout mouse by feeding a liquid diet; phenotype of the adult acetylcholinesterase deficient mouse

Acetylcholinesterase (AChE, EC3.1.1.7) functions in nerve impulse transmission, and possibly as a cell adhesion factor during neurite outgrowth. These functions predicted that a mouse with zero AChE activity would be unable to live. It was a surprise to find that AChE -/- mice were born alive and survived an average of 14 days. The emaciated appearance of AChE -/- mice suggested an inability to obtain sufficient nutrition and experiments were undertaken to increase caloric intake. Pregnant and lactating dams (+/-) were fed 11% high fat chow supplemented with liquid Ensure. AChE -/- pups were weaned early, on day 15, and fed liquid Ensure. Although nullizygous animals showed slow but steady weight gain with survival over 1 year (average 100 days), they remained small at all ages compared to littermates. They demonstrated delays in temperature regulation (day 22 vs. 15), eye opening (day 13 vs. 12), righting reflex (day 18 vs. 12), descent of testes (week 7-8 vs. 4), and estrous (week 15-16 vs. 6-7). Significant physical findings in adult AChE -/- mice included body tremors, abnormal gait and posture, absent grip strength, inability to eat solid food, pinpoint pupils, decreased pain response, vocalization, and early death caused by seizures or gastrointestinal tract ileus. Behavioral deficits included urination and defecation in the nest, lack of aggression, reduced pain perception, and sexual dysfunction. These findings support the classical role for AChE in nerve impulse conduction and further suggest that AChE is essential for timely physical development and higher brain function.

Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity

Central cholinergic systems are involved in a plethora of brain functions and are severely and selectively damaged in neurodegenerative diseases such as Alzheimer’s disease and dementia with Lewy bodies. Cholinergic dysfunction is treated with inhibitors of acetylcholinesterase (AChE) while the role of butyrylcholinesterase (BChE) for brain cholinergic function is unclear. We have used in vivo microdialysis to investigate the regulation of hippocampal acetylcholine (ACh) levels in mice that are devoid of AChE (AChE‐/‐ mice). Extracellular ACh levels in the hippocampus were 60‐fold elevated in AChE‐/‐ mice compared with wild‐type (AChE+/+) animals. In AChE‐/‐ mice, calcium‐free conditions reduced hippocampal ACh levels by 50%, and infusion of tetrodotoxin by more than 90%, indicating continuous ACh release. Infusion of a selective AChE inhibitor (BW284c51) caused a dose‐dependent, up to 16‐fold increase of extracellular ACh levels in AChE+/+ mice but did not change ACh levels in AChE‐/‐ mice. In contrast, infusion of a selective inhibitor of BChE (bambuterol) caused up to fivefold elevation of ACh levels in AChE‐/‐ mice, but was without effect in AChE+/+ animals. These results were corroborated with two other specific inhibitors of AChE and BChE, tolserine and bis‐norcymserine, respectively. We conclude that lack of AChE causes dramatically increased levels of extracellular ACh in the brain. Importantly, in the absence of AChE, the levels of extracellular ACh in the brain are controlled by the activity of BChE. These results point to a potential usefulness of BChE inhibitors in the treatment of central cholinergic dysfunction in which brain AChE activity is typically reduced.

Regulation of muscarinic acetylcholine receptor function in acetylcholinesterase knockout mice

Acetylcholinesterase (AChE) hydrolyzes acetylcholine to terminate cholinergic neurotransmission. Overstimulation of cholinergic receptors by excess acetylcholine is known to be lethal. However, AChE knockout mice live to adulthood, although they have weak muscles, do not eat solid food, and die early from seizures. We wanted to know what compensatory factors allowed these mice to survive. We had previously shown that their butyrylcholinesterase activity was normal and had not increased. In this report, we tested the hypothesis that AChE-/- mice adapted to the absence of AChE by downregulating cholinergic receptors. Receptor downregulation is expected to reduce sensitivity to agonists and to increase sensitivity to antagonists. Physiological response to the muscarinic agonists, oxotremorine (OXO) and pilocarpine, showed that AChE-/- mice were resistant to OXO-induced hypothermia, tremor, salivation, and analgesia, and to pilocarpine-induced seizures. AChE+/- mice had an intermediate response. The muscarinic receptor binding sites measured with [3H]quinuclinyl benzilate, as well as the protein levels of M1, M2, and M4 receptors measured with specific antibodies on Western blots, were reduced to be approximately 50% in AChE-/- brain. However, mRNA levels for muscarinic receptors were unchanged. These results indicate that one adaptation to the absence of AChE is downregulation of muscarinic receptors, thus reducing response to cholinergic stimulation.

Mice Treated with Chlorpyrifos or Chlorpyrifos Oxon Have Organophosphorylated Tubulin in the Brain and Disrupted Microtubule Structures, Suggesting a Role for Tubulin in Neurotoxicity Associated with Exposure to Organophosphorus Agents

Exposure to organophosphorus (OP) agents can lead to learning and memory deficits. Disruption of axonal transport has been proposed as a possible explanation. Microtubules are an essential component of axonal transport. In vitro studies have demonstrated that OP agents react with tubulin and disrupt the structure of microtubules. Our goal was to determine whether in vivo exposure affects microtubule structure. One group of mice was treated daily for 14 days with a dose of chlorpyrifos that did not significantly inhibit acetylcholinesterase. Beta-tubulin from the brains of these mice was diethoxyphosphorylated on tyrosine 281 in peptide GSQQY(281)RALTVPELTQQMFDSK. A second group of mice was treated with a single sublethal dose of chlorpyrifos oxon (CPO). Microtubules and cosedimenting proteins from the brains of these mice were visualized by atomic force microscopy nanoimaging and by Coomassie blue staining of polyacrylamide gel electrophoresis bands. Proteins in gel slices were identified by mass spectrometry. Nanoimaging showed that microtubules from control mice were decorated with many proteins, whereas microtubules from CPO-treated mice had fewer associated proteins, a result confirmed by mass spectrometry of proteins extracted from gel slices. The dimensions of microtubules from CPO-treated mice (height 8.7 +/- 3.1 nm and width 36.5 +/- 15.5 nm) were about 60% of those from control mice (height 13.6 +/- 3.6 nm and width 64.8 +/- 15.9 nm). A third group of mice was treated with six sublethal doses of CPO over 50.15 h. Mass spectrometry identified diethoxyphosphorylated serine 338 in peptide NS(338)NFVEWIPNNVK of beta-tubulin. In conclusion, microtubules from mice exposed to chlorpyrifos or to CPO have covalently modified amino acids and abnormal structure, suggesting disruption of microtubule function. Covalent binding of CPO to tubulin and to tubulin-associated proteins is a potential mechanism of neurotoxicity.

The butyrylcholinesterase knockout mouse as a model for human butyrylcholinesterase deficiency.

Butyrylcholinesterase (BChE) is an important enzyme for metabolism of ester drugs. Many humans have partial or complete BChE deficiency due to genetic variation. Our goal was to create a mouse model of BChE deficiency to allow testing of drug toxicity. For this purpose, we created the BChE knockout mouse by gene-targeted deletion of a portion of the BCHE gene (accession number M99492). The BChE–/– mouse had no BChE activity in plasma, but it had low residual butyrylthiocholine hydrolase activity in all other tissues attributed to carboxylesterase ES-10. The BChE–/– mouse had a normal phenotype except when challenged with drugs. Nicotinic receptor function as indicated by response to nicotine seemed to be normal in BChE–/– mice, but muscarinic receptor function as measured by response to oxotremorine and pilocarpine was altered. Heart rate, blood pressure, and respiration, measured in a Vevo imager, were similar in BChE+/+ and BChE–/– mice. Like BChE–/– humans, the BChE–/– mouse responded to succinylcholine with prolonged respiratory arrest. Bambuterol was not toxic to BChE–/– mice, suggesting it is safe in BChE–/– humans. Challenge with 150 mg/kg pilocarpine i.p., a muscarinic agonist, or with 50 mg/kg butyrylcholine i.p., induced tonicclonic convulsions and death in BChE–/– mice. This suggests that butyrylcholine, like pilocarpine, binds to muscarinic receptors. In conclusion, the BChE–/– mouse is a suitable model for human BChE deficiency.

The butyrylcholinesterase knockout mouse is obese on a high-fat diet

Butyrylcholinesterase (BChE) inactivates the appetite stimulating hormone octanoyl-ghrelin. The hypothesis was tested that BChE-/- mice would have abnormally high body weight and high levels of octanoyl-ghrelin. It was found that BChE-/- mice fed a standard 5% fat diet had normal body weight. However, BChE-/- mice fed a diet containing 11% fat became obese. Their obesity was not explained by increased levels of octanoyl-ghrelin, or by increased caloric intake, or by decreased exercise. Instead, a role for BChE in fat utilization was suggested.

Reduced acetylcholine receptor density, morphological remodeling, and butyrylcholinesterase activity can sustain muscle function in acetylcholinesterase knockout mice†

Nerve‐evoked contractions were studied in vitro in phrenic nerve–hemidiaphragm preparations from strain 129X1 acetylcholinesterase knockout (AChE−/−) mice and their wild‐type littermates (AChE+/+). The AChE−/− mice fail to express AChE but have normal levels of butyrylcholinesterase (BChE) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE−/− mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild‐type animals. In AChE−/− preparations, repetitive stimulation at frequencies of 20 and 50 Hz and at 200 and 400 Hz produced decremental muscle tensions; however, stimulation at 70 and 100 Hz resulted in little or no loss of tension during trains. Muscles from AChE+/+ mice maintained tension at all frequencies examined but exhibited tetanic fade after exposure to the selective AChE inhibitor 1,5‐bis(4‐allyldimethyl‐ammoniumphenyl)pentane‐3‐one (BW 284C51). The ability of diaphragm muscles from AChE−/− mice to maintain tension at 70 and 100 Hz suggests a partial compensation for impairment of acetylcholine (ACh) hydrolysis. Three mechanisms—including a reliance on BChE activity for termination of ACh action, downregulation of nicotinic acetylcholine receptors (nAChRs), and morphological remodeling of the endplate region—were identified. Studies of neuromuscular transmission in this model system provide an excellent opportunity to evaluate the role of AChE without complications arising from use of inhibitors. Muscle Nerve 30: 317–327, 2004

Glucocorticoid Mediation of Dietary Energy Restriction Inhibition of Mouse Skin Carcinogenesis

Dietary energy restriction (DER) inhibits carcinogenesis in numerous animal models. DER is a potent and reproducible inhibitor of two-stage mouse skin carcinogenesis when administered during the promotion phase. Previous research demonstrated that adrenalectomy abolished cancer prevention by food restriction. Several lines of evidence suggest that glucocorticoid elevation in the DER mouse mediates the prevention of skin cancer. Our research tested the hypothesis that elevated glucocorticoid hormone activates the glucocorticoid receptor (GR) and that this activated receptor interferes with the activator protein-1 (AP-1) transcription factor. Induction of AP-1 by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is essential to tumor promotion. We have been unable to demonstrate elevated activated GR in the epidermis of the DER mouse, perhaps because only indirect strategies have been possible with the use of epidermis from DER mice. However, DER blocked the induction of AP-1 and c-jun, a constituent protein of AP-1, in the epidermis of mice. Current studies are focused on the inhibition of signaling down the MAP-1/Raf-1 kinase pathway that leads to induction of constituent proteins of AP-1, including c-Jun. Although several pathways lead to the induction of AP-1 transcriptional activity, the MAP-1/Raf-1 pathway can be activated by protein kinase C (PKC); previous studies from our laboratory demonstrated an inhibition of PKC activity and a reduction in selected isoforms of PKC in the epidermis of the DER mouse. Our current working hypothesis is that elevated glucocorticoid hormone in the DER mouse reduces the amount and activity of PKC isoforms important in the activation of MAP-1/Raf-1 kinase pathway. We propose that this results in attenuation in the induction of the AP-1 transcription factor by TPA. Because AP-1 induction by TPA is obligatory for mouse skin promotion, we propose this as an essential component of the mechanism of DER prevention of mouse skin carcinogenesis.

Altered hippocampal muscarinic receptors in acetylcholinesterase-deficient mice

A primary therapeutic strategy for Alzheimers disease includes acetylcholinesterase (AChE) inhibitors with the goal of enhancing cholinergic transmission. Stimulation of muscarinic acetylcholine receptors (mAChRs) by elevated levels of ACh plays a role in the effects of AChE inhibitors on cognition and behavior. However, AChE inhibitors only demonstrate modest symptomatic improvements. Chronic treatment with these drugs may cause mAChR downregulation and consequently limit the treatment efficacy. AChE knockout (−/−) mice were utilized in this study as a model for investigating the effects of selective, complete, and chronic diminished AChE activity on mAChR expression and function. In AChE −/− mice, the M1, M2, and M4 mAChRs showed strikingly 50 to 80% decreased expression in brain regions associated with memory. In addition, mAChRs showed decreased presynaptic, cell surface, and dendritic distributions and increased localization to intracellular puncta. Furthermore, mAChR agonist–induced activation of extracellular signal–regulated kinase, a signaling pathway associated with synaptic plasticity and amyloidogenesis, is diminished in the hippocampus and cortex of AChE −/− mice. Therefore, chronic diminished ACh metabolism produces profound effects on mAChR expression and function. The alterations of mAChRs in AChE −/− mice suggest that mAChR downregulation may contribute to the limited efficacy of AChE inhibitors in Alzheimers disease treatment. Ann Neurol 2003;53:788–796

Gene-Delivered Butyrylcholinesterase Is Prophylactic against the Toxicity of Chemical Warfare Nerve Agents and Organophosphorus Compounds

Gene delivery using an adenoviral system has been effective in introducing therapeutic proteins in vitro and in vivo. This study tested the feasibility of using adenovirus to deliver clinically relevant amounts of butyrylcholinesterase (BChE), a proven bioscavenger of nerve agents. The adenovirus construct expressed full-length mouse BChE. Mice were injected with a single dose of adenovirus (1.5 × 1010 infectious units) in the tail vein; plasma was collected through day 11 and assayed for BChE activity. Maximum activity, representing a 300- to 3400-fold increase over baseline, was found on day 4. Expression levels returned to baseline by day 10. Nondenaturing gel electrophoresis showed the recombinant BChE was a dimer that could be converted to tetramers by addition of polyproline. The toxic compounds chosen for protection studies were positively charged organophosphorus agents, echothiophate, and O-ethyl-S-2-N,N-diisopropylaminoethyl methylphosphonothiolate (VX). Mice containing elevated blood levels of BChE (300- to 3,000-fold over the control mice) were challenged with incremental doses of echothiophate or VX. Mice showed no signs of toxicity and were protected from up to 30× LD50 dose of echothiophate and 5× LD50 dose of VX. A good correlation was observed between tolerated echothiophate dose and plasma BChE levels at time of challenge. The absolute increases in levels of circulating BChE and the sustained nature of the response resulted in a very high enzyme concentration, deemed critical in acute toxicity (5× LD50 or more) scenarios. These results suggest that gene-delivered BChE is a prophylactic and affords protection equivalent to that of a multimilligram injection of the same.