Gene-delivered butyrylcholinesterase is prophylactic against the toxicity of chemical warfare nerve agents and organophosphorous

ABSTRACT 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 ϫ 10 10 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 30ϫ LD 50 dose of echothiophate and 5ϫ LD 50 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 (5ϫ LD 50 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

Evidence for Nonacetylcholinesterase Targets of Organophosphorus Nerve Agent: Supersensitivity of Acetylcholinesterase Knockout Mouse to VX Lethality

The possibility that organophosphate toxicity is due to inhibition of targets other than acetylcholinesterase (AChE, EC 3.1.1.7) was examined in AChE knockout mice. Mice (34–55 days old) were grouped for this study, after it was determined that AChE, butyrylcholinesterase (BChE), and carboxylesterase activities had reached stable values by this age. Mice with 0, 50, or 100% AChE activity were treated subcutaneously with the nerve agent VX. The LD50 for VX was 10 to 12 µg/kg in AChE-/-, 17 µg/kg in AChE+/-, and 24 µg/kg in AChE+/- mice. The same cholinergic signs of toxicity were present in AChE-/- mice as in wild-type mice, even though AChE-/- mice have no AChE whose inhibition could lead to cholinergic signs. Wild-type mice, but not AChE-/- mice, were protected by pretreatment with atropine. Tissues were extracted from VX-treated and untreated animals and tested for AChE, BChE, and acylpeptide hydrolase activity. VX treatment inhibited 50% of the AChE activity in brain and muscle of AChE+/+ and +/- mice, 50% of the BChE activity in all three AChE genotypes, but did not significantly inhibit acylpeptide hydrolase activity. It was concluded that the toxicity of VX must be attributed to inhibition of nonacetylcholinesterase targets in the AChE-/- mouse. Organophosphorus ester toxicity in wild-type mice is probably due to inhibition or binding to several proteins, only one of which is AChE

Impaired formation of the inner retina in an AChE knockout mouse results in degeneration of all photoreceptors.

Blinding diseases can be assigned predominantly to genetic defects of the photoreceptor/pigmented epithelium complex. As an alternative, we show here for an acetylcholinesterase (AChE) knockout mouse that photoreceptor degeneration follows an impaired development of the inner retina. During the first 15 postnatal days of the AChE-/- retina, three major calretinin sublaminae of the inner plexiform layer (IPL) are disturbed. Thereby, processes of amacrine and ganglion cells diffusely criss-cross throughout the IPL. In contrast, parvalbumin cells present a nonlaminar IPL pattern in the wild-type, but in the AChE-/- mouse their processes become structured within two 'novel' sublaminae. During this early period, photoreceptors become arranged regularly and at a normal rate in the AChE-/- retina. However, during the following 75 days, first their outer segments, and then the entire photoreceptor layer completely degenerate by apoptosis. Eventually, cells of the inner retina also undergo apoptosis. As butyrylcholinesterase (BChE) is present at a normal level in the AChE-/- mouse, the observed effects must be solely due to the missing AChE. These are the first in vivo findings to show a decisive role for AChE in the formation of the inner retinal network, which, when absent, ultimately results in photoreceptor degeneration

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 GSQQY281RALTVPELTQQMFDSK. 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 NS338NFVEWIPNNVK 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

REDUCED ACETYLCHOLINE RECEPTOR DENSITY, MORPHOLOGICAL REMODELING, AND BUTYRYLCHOLINESTERASE ACTIVITY CAN SUSTAIN MUSCLE FUNCTION IN ACETYLCHOLINESTERASE KNOCKOUT MICE

The vertebrate neuromuscular junction is designed for rapid transmission of excitatory signals for initiation of muscle contraction.5 Among the features responsible for the high throughput of this synapse are the close proximity of the presynaptic and postsynaptic membranes,10 the direct coupling of acetylcholine (ACh) binding to the opening of the ion channel associated with the nicotinic acetylcholine receptor (nAChR),27 the brief open time of this channel,21,27 and the presence of cholinesterase (ChE) for hydrolysis of ACh.21,30 At the endplate, there are two distinct ChEs for transmitter hydrolysis: acetylcholinesterase (EC 3.1.1.7, AChE) and butyrylcholinesterase (EC 3.1.1.8, BChE).33 Both enzymes can exist in a multisubunit, collagen-tailed form with selective localization at the endplate basallamina.33 Because of its superior catalytic activity for ACh hydrolysis, AChE is the dominant enzyme, whereas the role of BChE is generally evident only after AChE is inhibited.3,4 Inhibition of ChE results in a progressive accumulation of ACh, especially during periods of repetitive stimulation, leading to desensitization of nAChRs and consequent muscle weakness.12,17 Under this condition, transmitter persists beyond its normal lifetime and is slowly removed from the endplate region by diffusion.21,30 Diffusion is impeded in part by morphological barriers, such as the apposition of the nerve terminal to the postjunctional membrane,5,10 and by the high density of postjunctional nAChRs.21,22,25 If ChE is inhibited pharmacologically or removed by collagenase treatment, repeated binding to nAChR makes diffusional loss of ACh slow and inefficient.21,22,30 The influence of nAChRs on retention of transmitter was termed “buffered diffusion” by Katz and Miledi21 and accounts for findings that elimination of ACh is considerably slower than that expected for free diffusion. 30 Inhibitors of ChE are highly toxic, producing incapacitation and death within minutes.28 The cause of death is complex, involving loss of central respiratory drive,6,29 bronchospasm,1,2 and the inability of the diaphragm muscle to sustain tetanic tension. 19 Because most ChE inhibitors show little selectivity between AChE and BChE, and may have direct actions unrelated to ChE inhibition, it is difficult to establish the role of AChE activity in neuromuscular transmission. To overcome this difficulty, we studied twitch and tetanic tensions in diaphragm muscles from AChE knockout (AChE-/-) mice that fail to express AChE but do contain normal levels of BChE.7,24,36 Stimulation of the phrenic nerve in isolated diaphragm preparations from AChE-/- mice revealed large single twitches and sustained tetanic tensions at 70 and 100 Hz. These findings suggest that, over a limited frequency range, diaphragm muscles from AChE-/- mice are able to compensate for the loss of AChE activity. An understanding of these adaptive mechanisms is expected to provide insight on protection strategies that may be effective against the toxic actions of ChE inhibitors such as the highly lethal nerve agents