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Human Molecular Genetics Advance Access originally published online on October 7, 2004
Human Molecular Genetics 2004 13(23):2947-2957; doi:10.1093/hmg/ddh320
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Human Molecular Genetics, Vol. 13, No. 23 © Oxford University Press 2004; all rights reserved

A mouse model of AChR deficiency syndrome with a phenotype reflecting the human condition

Judy Cossins, Richard Webster, Susan Maxwell, Georgina Burke, Angela Vincent and David Beeson*

Neurosciences Group, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford OX3 9DS, UK

Received July 30, 2004; Accepted September 26, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The two subtypes of mammalian muscle nicotinic acetylcholine receptors (AChR) are generated by the substitution of the {varepsilon} (adult) subunit for the {gamma} (fetal) subunit within the AChR pentamer. Null mutations of the adult AChR {varepsilon}-subunit gene are the most common cause of the AChR deficiency syndrome. This is a disorder of neuromuscular transmission characterized by non-progressive fatigable muscle weakness present throughout life. In contrast with the human disorder, mice with AChR {varepsilon}-subunit null mutations die between 10 and 14 weeks of age. We generated transgenic mice that constitutively express the human AChR {gamma}-subunit in an AChR {varepsilon}-subunit ‘knock-out’ background. These mice, in which neuromuscular transmission is mediated by fetal AChR, live well into adult life but show striking similarities to human AChR deficiency syndrome. They display fatigable muscle weakness, reduced miniature endplate potentials and endplate potentials, reduced motor endplate AChR number and altered endplate morphology. Our results illustrate how species differences in the control of ion-channel gene expression may affect disease phenotype, demonstrate that expression of adult AChR subtype is not essential for long-term survival, and suggest that in patients with AChR deficiency syndrome, up-regulation of the {gamma}-subunit could be a beneficial therapeutic strategy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Many genetic disorders have been reproduced in transgenic mice by introduction of the relevant gene defect, but it is common to find that the mouse phenotype poorly reflects the severity and/or variability of the human disease. Acetylcholine receptor (AChR) deficiency syndrome is a typical example. In mammals, neuromuscular signal transmission is mediated through ‘fetal’ or ‘adult’ forms of the AChR (1). The fetal AChR has a subunit composition of {alpha}2ß{gamma}{delta}, whereas at the motor endplate of adult muscle the {gamma}-subunit is replaced by an {varepsilon}, {alpha}2ß{delta}{varepsilon} (2). In AChR deficiency, a recessively inherited congenital myasthenic syndrome (CMS), a marked reduction in the number of AChRs at the motor endplate can be demonstrated by labelled {alpha}-bungarotoxin ({alpha}-BuTx) (3). The majority of mutations that underlie this syndrome are located at the AChR {varepsilon}-subunit gene locus, and most patients harbour either homozygous or compound heterozygous null mutations (reviewed in 4,5) that predict a null phenotype. However, although the patients suffer from early infancy with generalized fatigable muscle weakness, ptosis, impaired eye movements and delayed motor milestones, most have a normal lifespan.

Transgenic mice with targeted disruption of the {varepsilon}-subunit gene have been generated by two separate laboratories (6,7). In each report, the homozygous mutant mice appear normal for the first few weeks of life, but subsequently become less active, fail to put on weight and die between 8 and 14 weeks of age. The deterioration in muscle strength is associated with loss of endplate AChR, so that when the mutant mice are beginning to die the endplates have ~5% of wild-type AChR numbers (7). The severity of the phenotype of these {varepsilon}-subunit knock-out mice is clearly different to that of the phenotype of AChR deficiency syndrome in humans.

This may be because of species differences in AChR {gamma}-subunit expression: in mice, transcription of the AChR {gamma}-subunit mRNA is difficult to detect in adult muscle, even by PCR, whereas in mature human muscle the {gamma}-subunit mRNA is readily detectable (8). It has been proposed that incorporation of the AChR {gamma} (fetal-specific) subunit into the endplate AChR pentamers enables the CMS patients with two {varepsilon}-subunit null alleles to survive (9,10). However, in situ hybridization experiments suggest that in AChR deficiency syndrome patients there is no transcriptional up-regulation of fetal AChR (11). We hypothesize, therefore, that fetal AChR is provided by the residual low levels of {gamma}-subunit mRNA normally transcribed at human subsynaptic and extrasynaptic nuclei.

In order to test this hypothesis, and to assess the extent to which the fetal AChR can substitute for adult AChR at the mature neuromuscular junction, we generated transgenic mice lacking the AChR {varepsilon}-subunit that constitutively express the human AChR {gamma}-subunit.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of transgenic mice expressing human AChR {gamma}-subunit in an {varepsilon}-subunit null background
A transgene cassette was constructed that contained a cDNA clone containing the full human AChR {gamma}-subunit coding sequence (12) ligated 3' to the human skeletal {alpha}-actin promoter (13) (Fig. 1A). The human AChR {gamma}-subunit cDNA was chosen to enable easy distinction between expression of the transgene and endogenous expression of the mouse AChR {gamma}-subunit. Transgenic mice were identified by PCR using human AChR {gamma}-subunit-specific primers, and expression of human {gamma}-subunit mRNA was confirmed by RT–PCR for three different regions of the human {gamma}-subunit cDNA (Fig. 1B). The {gamma}19 mice, which harbour the human AChR {gamma}-subunit expression cassette (h{gamma}+), were bred with heterozygous {varepsilon}+/– mice and h{gamma}+{varepsilon}+/– progeny identified. These mice were then crossed (h{gamma}+{varepsilon}+/–xh{gamma}+{varepsilon}+/–) and the resulting litters genotyped to identify mice expressing the human AChR {gamma}-subunit transgene in an {varepsilon}-subunit null background (h{gamma}+{varepsilon}–/–) (Fig. 1C, Table 1). Heterozygous {varepsilon}+/– littermates, which show no phenotypic defects (6,7), were used as controls.





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  		Figure 1. (A) Representation of the transgene cassette containing the human {alpha}-actin skeletal muscle (HSA) promoter and the human muscle AChR {gamma}-subunit cDNA. (B) Expression of the human AChR {gamma}-subunit mRNA in the transgenic mouse line {gamma}19. RNA from a mouse harbouring the transgene cassette was subjected to RT–PCR using three separate human muscle AChR {gamma}-subunit-specific oligonucleotide primer pairs (see Materials and Methods), and mouse AChR {alpha}-subunit-specific primers. The products were size-fractionated on a 2% agarose gel, stained with ethidium bromide and visualized under UV light. (C) Genotyping of the progeny from h{gamma}+{varepsilon}+/–xh{gamma}+{varepsilon}+/– mice. Mouse genomic DNA was used as a template for PCR amplifications designed to detect the human AChR {gamma}-subunit cDNA (h{gamma}), the neomycin resistance gene that disrupts the mouse AChR {varepsilon}-subunit gene (neo), and the mouse AChR {varepsilon}-subunit gene ({varepsilon}). PCR products were size-fractionated on a 2% agarose gel, stained with ethidium bromide and visualized under UV light. Example genotypes are shown.

 

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  		Table 1. Genotyping of the transgenic mice
 
Expression of the human AChR {gamma}-subunit mRNA in mouse muscle was confirmed by northern blot analysis at 8 weeks of age. Human AChR {gamma}-subunit mRNA was only detected in the h{gamma}+ mice, and within the h{gamma}+ mice was found in muscle but not in brain or liver, confirming tissue-specific expression from the muscle {alpha}-actin promoter (Fig. 2A and B). Translation of the mRNA and incorporation of the human {gamma}-subunit into the 125I-{alpha}-BuTx-labelled AChR was verified by immunoprecipitation of 125I-{alpha}-BuTx-binding sites from muscle extracts at 4 weeks of age using the human {gamma}-subunit-specific monoclonal antibody, mab C9 (Fig. 2C). Mab C9 precipitated 125I-{alpha}-BuTx-bound AChR from muscle extracts of h{gamma}+ mice but not from {varepsilon}+/– or {varepsilon}–/– mice, demonstrating the species specificity of C9 and that it retains its ability to bind the human {gamma}-subunit when co-expressed with mouse {alpha}-, ß- and {delta}-subunits.




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  		Figure 2. Analysis of expression of the human AChR {gamma}-subunit in transgenic mice. (A) Northern blot of total RNA extracted from mouse leg muscle, probed with either 32P-labelled human AChR {gamma}-subunit cDNA (top panel) or 32P-labelled mouse AChR {alpha}-subunit cDNA as a control (lower panel). (B) Northern blot analysis of the expression of human AChR {gamma}-subunit mRNA in muscle, brain and liver from h{gamma}+ mice. GAPDH was used as a positive control for the RNA (lower panel). (C) Immunoprecipitation of 125I-{alpha}-BuTx-labelled AChR using a monoclonal antibody specific for the human {gamma}-subunit, mab C9. Aliquots of Triton X-100 extracts from leg muscle of 4-week-old {varepsilon}+/–, {varepsilon}–/– and h{gamma}+{varepsilon}–/– mice were labelled with 125I-{alpha}-BuTx and used in immunoprecipitations. Background binding after preincubation of parallel samples with an excess of cold {alpha}-BuTx has been subtracted.

 
Phenotype of h{gamma}+{varepsilon}–/– transgenic mice
As reported previously (7) the mice homozygous for the AChR {varepsilon}-subunit null mutation appear externally normal for the first few weeks after birth, but by ~4 weeks of age begin to lose weight, become less active, acquire a waddling gait and begin to die between 8 and 10 weeks of age. In contrast, the h{gamma}+{varepsilon}–/– mice appear externally normal for the first few weeks after birth, show only slight gain in weight after 4 weeks, less than that of the {varepsilon}+/– littermates (Fig. 3), but continue to move around the cage freely although in general are less active than {varepsilon}+/– littermates. Growth curves were similar for male and female mice. Both the {varepsilon}–/– and the h{gamma}+{varepsilon}–/– mice spontaneously emit ‘whistling’ or ‘bird-like chattering’ sounds as reported previously for {varepsilon}–/– mice (14) or acetylcholinesterase –/– mice (15). Shortly after 8 weeks of age, it is necessary to sacrifice all {varepsilon}–/– mice, but the h{gamma}+{varepsilon}–/– mice have been kept for more than 9 months without deterioration in their condition.



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  		Figure 3. Growth curve of male mice. {varepsilon}+/– (open triangle), {varepsilon}–/– (open square) and h{gamma}+{varepsilon}–/– (closed circle) mice were weighed up to 10 weeks post-weaning and the average weight at each time point for each genotype calculated.

 
Evaluation of muscle strength
Muscle strength of h{gamma}+{varepsilon}–/–, {varepsilon}–/– and {varepsilon}+/– mice were evaluated at 4 and 8 weeks of age, by blinded tests, using an inverted screen test for sustained effort and a forelimb weight lift test for short-term strength (Fig. 4A and B). Compared with the {varepsilon}+/– heterozygotes, the {varepsilon}–/– mice were severely impaired at 4 weeks in both tests, and by 8 weeks, the three remaining mice were barely able to perform the tests (P-values are given in Table 2). In contrast, the h{gamma}+{varepsilon}–/– mice performed well on both tests at 4 and 8 weeks, although their values were not in general as good as the heterozygote controls (Fig. 4A and B). Thus, at 4 weeks, despite the lack of obvious phenotypic differences between the {varepsilon}–/– and the h{gamma}+{varepsilon}–/– mice, both tests showed that the h{gamma}+{varepsilon}–/– were much stronger, and were able to perform almost as well as the {varepsilon}+/– mice, particularly on the short-term weight lifting task. Strikingly, the h{gamma}+{varepsilon}–/– mice showed considerable heterogeneity in their muscle strength as indicated by these two tests.



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  		Figure 4. Mouse muscle strength tested at 4 and 8 weeks of age. (A) Inverted screen test. The length of time that each mouse could hold on upside-down to a wire mesh screen was recorded, with an endpoint of 2 min (for scoring system, see Materials and Methods). At both ages, the medians for all three genotypes are significantly different from each other (Table 2). (B) Forelimb weight lift test. At 4 weeks of age the {varepsilon}–/– mice were significantly weaker than {varepsilon}+/– mice and h{gamma}+{varepsilon}–/– mice. The number of mice for both tests were: at 4 weeks for {varepsilon}+/–, n=12; {varepsilon}–/–, n=9; h{gamma}+{varepsilon}–/–, n=18; and at 8 weeks for {varepsilon}+/–, n=6; {varepsilon}–/–, n=3; h{gamma}+{varepsilon}–/–, n=13.

 

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  		Table 2. P-Values in the comparison of genotypes tested for muscle strength
 
Electrophysiology
To explore further the improved function of the h{gamma}+{varepsilon}–/– mice, we analyzed the amplitudes of the endplate potentials (EPPs) and miniature endplate potentials (MEPPs) at the motor endplates. We first confirmed that the amplitudes of the EPPs (29.2±1.70 mV versus 27.1±0.68 mV) and MEPPs (1.40±0.08 mV versus 1.16±0.04 mV) for the heterozygous {varepsilon}+/– mice were not different to wild-type mice. We then compared {varepsilon}+/–, {varepsilon}–/– and h{gamma}+ {varepsilon}–/– mice. Examples of recordings from these three groups of experimental mice at 8 weeks are shown in Figure 5, and a summary of the results in Table 3. Importantly, it was noticeably more difficult to find muscle fibres with EPPs with fast rise times in the {varepsilon}–/– compared with the other two mouse groups, and in some cases the MEPPs were undetectable against the background noise of the recording system. The loss of MEPPs in the noise of the recording system is known to be a problem when trying to obtain a true mean value for MEPP amplitudes, and when calculating the quantal content from EPP/MEPP ratios. The results from the {varepsilon}–/– mice, therefore, are likely to be overestimates.




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  		Figure 5. (A) Spontaneous miniature endplate recordings from phrenic nerve/hemi-diaphragm preparations of 8-week-old mice. Upper panel shows example traces from MEPP recordings of {varepsilon}+/–, {varepsilon}–/– and h{gamma}+{varepsilon}–/– mice, with signal averaged from up to 30 records. Scale bars refer to all three traces. Lower panel shows averaged MEPP from each endplate (acquired from up to 30 individual records) in each group. Horizontal bars indicate mean±SEM of all endplates recorded in that group. (B) Evoked endplate recordings from phrenic nerve/hemi-diaphragm preparation of 8-week-old mice. Upper panel shows example traces from EPP recordings of {varepsilon}+/–, {varepsilon}–/– and h{gamma}+{varepsilon}–/– mice, signal averaged from up to 30 records. Scale bars refer to all three traces. Lower panel shows averaged EPP from each endplate (acquired from up to 30 individual records) in each group. Horizontal bars indicate mean±SEM of all endplates recorded in that group.

 

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  		Table 3. MEPPs and EPPs recorded from mouse hemi-diaphragms at 8 weeks of age
 
The amplitudes of both the EPPs and MEPPs were significantly reduced in h{gamma}+{varepsilon}–/– and {varepsilon}–/– mice compared with {varepsilon}+/– mice (Fig. 5A and B), and the EPP amplitude was significantly greater in h{gamma}+{varepsilon}–/– mice compared with {varepsilon}–/– mice (P<0.001) (Table 3). However, the MEPP amplitudes were not very different, although both were reduced compared with the {varepsilon}+/– controls. This is likely to be because of the difficulties in identifying reduced amplitude MEPPs as outlined above, as MEPPs were only recordable in 14 of the 30 endplates examined from the three {varepsilon}–/– mice available for study at 8 weeks, compared with 46 of the 48 endplates examined from the h{gamma}+{varepsilon}–/– mice (Fisher exact test P<0.001). There were no significant differences in quantal content between {varepsilon}+/–, {varepsilon}–/– or h{gamma}+{varepsilon}–/– mice (data not shown).

Quantification of endplate AChR
To see how well AChR numbers correlated with the improved muscle strength in the h{gamma}+{varepsilon}–/– mice, we measured endplate 125I-{alpha}-BuTx binding in mouse diaphragms. To be able to compare between different diaphragms, we expressed the binding as cpm/mg/cm length of muscle fibres (14). The values in the {varepsilon}+/– mice represent ~5x107 {alpha}-BuTx binding sites per endplate (unpublished data). At both 4 and 8 weeks of age, endplate binding for both h{gamma}+{varepsilon}–/– and {varepsilon}–/– was reduced to <25% of control levels (Fig. 6), consistent with the AChR deficiency phenotype of the mice.



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  		Figure 6. 125I-{alpha}-BuTx binding to endplate regions from hemi-diaphragms prepared from 4 or 8-week-old mice (mean±SD, n=6, except for {varepsilon}–/– at 8 weeks, n=4).

 
Distribution of AChR at endplates
To explore further the amount and nature of the AChRs at the endplates, we labelled diaphragms with tetramethylrhodamine {alpha}-BuTx (Fig. 7A and B). At 4 weeks, the fluorescent signal from h{gamma}+{varepsilon}–/– and {varepsilon}–/– was less intense than the {varepsilon}+/– control littermates. Whereas AChR at endplates from control {varepsilon}+/– were evenly labelled and ‘pretzel-shaped’, the endplate AChR of {varepsilon}–/– mice often appeared more patchy with variable regions of high and low AChR density, the highest density tending to be at the outer edge of the endplate region. Endplate AChR from h{gamma}+{varepsilon}–/– mice showed a less well formed ‘pretzel shape’ and tended to appear as several smaller patches of intense labelling. By 8 weeks, the signal from {varepsilon}–/– mice was very difficult to detect (data not shown), whereas signal from h{gamma}+{varepsilon}–/– again often appeared as a number of small unconnected patches spreading along the muscle fibre (upper panel) or occasionally had the fragmented appearance seen in the 4 week {varepsilon}–/– mice (lower panel).




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  		Figure 7. Motor endplates on mouse diaphragm muscle labelled with tetramethylrhodamine-conjugated {alpha}-BuTx. (A) Examples of endplates from {varepsilon}+/–, {varepsilon}–/– and h{gamma}+{varepsilon}–/– mice at 4 weeks and (B) of {varepsilon}+/– and h{gamma}+{varepsilon}–/– mice at 8 weeks. Scale bars represent 20 µm.

 
Location of the human AChR {gamma} subunits in mouse muscle
Finally, to visualize incorporation of the human {gamma}-subunit into the endplates, we used a polyclonal rabbit antibody to AChR {alpha}-subunit that binds to mouse AChR, and a monoclonal antibody specific for human AChR {gamma}-subunit (C9) on sections from 4 or 8-week-old mice (Fig. 8A and B). This monoclonal antibody does not precipitate mouse fetal AChR (17) and does not bind AChR in the {varepsilon}–/– mice (Fig. 2C).





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  		Figure 8. Immunolabelling of mouse endplate. Cross-sections of (A) 4-week-old or (B) 8-week-old, h{gamma}+{varepsilon}–/– mouse gastrocnemius muscle labelled with anti-AChR {alpha}-subunit polyclonal antibody ({alpha}, left panels); anti-human AChR {gamma}-subunit monoclonal antibody, C9 ({gamma}, centre panels); and merged images (right panels). (C) Immunolabelling of gastrocnemius muscle from an 8-week-old h{gamma}+{varepsilon}+/– mouse. Scale bars represent 20 µm.

 
Despite the fact that the human AChR {gamma}-subunit mRNA under the actin promoter is likely to be expressed evenly at nuclei along the length of the muscle fibre, expression of the human AChR {gamma}-subunit was concentrated at the endplates at 4 and 8 weeks in the h{gamma}+{varepsilon}–/– mice and there was little evidence of fetal AChR at other sites. In contrast, in heterozygous mice that also harboured the transgene (h{gamma}+{varepsilon}+/–) there was no apparent expression of human AChR {gamma}-subunit at endplates, suggesting that in these mice the human {gamma}-subunit is out-competed by endogenous mouse {varepsilon}-subunits derived from mRNA synthesized by the subsynaptic nuclei (Fig. 8C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Transgenic AChR {varepsilon}-subunit knock-out mice have a severe phenotype with death around 8–14 weeks of age, whereas the human disease associated with {varepsilon}-subunit null mutations, AChR deficiency syndrome, is far less severe. We have generated a transgenic mouse model that shows striking similarities to the human disease. By introducing a transgene expressing the human {gamma}-subunit into the AChR {varepsilon}-subunit null background, we have produced mice that are less robust than wild-type or heterozygous {varepsilon}+/– mice, show weakness and fatigue, with reduced AChR numbers, and reduced MEPP and EPP amplitudes at their endplates, but survive well into adult life. Their muscle AChRs have incorporated human {gamma}-subunits, as shown by immunoprecipitation and immunofluorescence. Thus, our findings also indicate that neuromuscular transmission can be mediated by fetal AChR throughout life, and that adult AChR is not crucial for long-term survival.

The expression profile of fetal AChRs shows distinct species variation between humans and mice. In mice, there are some differences in the switch from {gamma}- to {varepsilon}-subunit-containing AChR in different muscle types: in fast-twitch muscle the switch is essentially complete by P17, but in the predominantly slow-twitch soleus muscle it is delayed for an additional week (18). Activity-dependent down-regulation of AChR {gamma}-subunit mRNA (19) is efficient, and the presence of {gamma}-subunit mRNA in adult mouse muscle is difficult to detect even by RT–PCR. Moreover, in mice with targeted disruption of the {varepsilon}-subunit gene there appears to be no significant up-regulation of the {gamma}-subunit mRNA despite the reduced activity associated with endplate AChR numbers falling to <5% of wild-type (7). Consequently, mice homozygous for {varepsilon}-subunit null alleles die between 8 and 14 weeks of age owing to lack of endplate AChR (6,7). In humans, the {gamma} to {varepsilon} switch is thought to be largely completed before birth and the {gamma}-subunit was not detected in AChR at endplates after 31 weeks gestation (20). However, {gamma}-subunit mRNA is readily detectable in synaptic and extrasynaptic regions of adult human muscle by both RT–PCR and RNase protection assays (8). Nevertheless, in accordance with the results from the {varepsilon}-subunit knock-out mice, we could detect no evidence of {gamma}-subunit mRNA up-regulation at the endplates of AChR deficiency patients (11). We therefore designed our {gamma}-subunit cDNA transgene so that it is driven by the human {alpha}-actin promoter. This should result in synthesis of {gamma}-subunit mRNA from nuclei along the length of both fast- and slow-twitch fibres (11), although at lower levels in slow-twitch fibres (21).

The primary pathogenic feature of AChR deficiency syndrome is a severe reduction in endplate AChR numbers, which are reduced to between 10 and 30% of normal values (3,22). The disorder is not progressive. These features are recreated in the h{gamma}+{varepsilon}–/– mice, in which endplate AChR number is found to be ~20% of {varepsilon}+/– or wild-type mice at 4 weeks of age, and remain at this level as the animals age. The reduced AChR number probably results from low expression of the human AChR {gamma}-subunit mRNA as, although northern blots showed mouse {alpha}-subunit mRNA and human AChR {gamma}-subunit mRNA at roughly similar levels, expression of the {alpha}-subunit mRNA would be concentrated at the subsynaptic nuclei, whereas expression of the human AChR {gamma}-subunit mRNA is expected to occur throughout the muscle fibre. Thus, the expression of the human AChR {gamma}-subunit mRNA from subsynaptic nuclei is likely to be severely reduced when compared with mRNA encoding the mouse AChR {alpha}-, ß- or {delta}-subunits, as it is in human muscle (11). Moreover, we were unable to detect human AChR {gamma}-subunits at endplates of h{gamma}+{varepsilon}+/– (Fig. 8C), again suggesting that there is an excess of {varepsilon} over {gamma}.

Functional neuromuscular transmission was achieved in adult h{gamma}+{varepsilon}–/– mice owing to the incorporation of the human {gamma}-subunit into endplate receptors. We do not know whether the use of a promoter specific for subsynaptic mRNA expression (23), such as the AChR {alpha}-subunit gene promoter, would have resulted in normal numbers of fetal AChR and a near normal phenotype. Similarly, it is not evident how the different biophysical properties of fetal AChRs might affect the phenotype. Our aim was to provide a model of AChR deficiency syndrome. The {gamma}-subunits incorporated into the endplate receptors may be derived purely from sub-synaptic mRNA expression or there may be active recruitment of {gamma}-subunits from perijunctional regions. The images of endplates for h{gamma}+{varepsilon}–/– mice (Fig. 7A and B, upper panels for h{gamma}+{varepsilon}–/–) suggest some terminal sprouting (24) and recruitment of {gamma}-subunits into elongated regions of distinct small high-density patches along the muscle fibre.

The muscle strength of the h{gamma}+{varepsilon}–/– mice was assessed using both a forelimb strength test and an inverted screen grip test. The inverted screen test, which gives a measure of muscle fatigability, proved reliable and highly effective in differentiating between the three genotypes {varepsilon}+/–, {varepsilon}–/– and h{gamma}+{varepsilon}–/–. Estimates both of the safety margin for neuromuscular transmission (25) and analysis of patients with myasthenia gravis (26) suggest that muscle weakness is likely to be apparent when receptor numbers fall below 30% of normal. Similarly in AChR deficiency syndrome, levels of endplate receptors are present between 10 and 30% of control values (4,22). In accordance with these estimates, muscle weakness was apparent in h{gamma}+{varepsilon}–/– mice (P<0.0001) at 4 and 8 weeks of age when diaphragm receptor levels were ~20% of {varepsilon}+/– controls.

Electrophysiological recordings comparing {varepsilon}+/+ and {varepsilon}+/– mice indicated normal EPP and MEPP amplitudes for heterozygous mice, consistent with previous reports (6), and with the lack of any phenotype in humans heterozygous for AChR {varepsilon}-subunit null alleles (9,11). In theory, the decreased amplitude of MEPP and EPP in {varepsilon}–/– and h{gamma}+{varepsilon}–/– mice could be the result of changes in muscle fibre input resistance. However, as results from these recordings correspond closely with the {alpha}-BuTx binding studies, and quantal content did not alter, it is highly likely that the reduced MEPP and EPP amplitudes in the {varepsilon}–/– and h{gamma}+{varepsilon}–/– mice demonstrate impaired neuromuscular transmission caused by reduced AChR density and number.

Previously we surmised that persistent low-level expression of the AChR {gamma}-subunit, which is present in normal human muscle as well as in patients with AChR deficiency syndrome, is sufficient to enable patients with {varepsilon}-subunit null alleles to survive (11). This animal model, in which there is non-progressive fatigable muscle weakness resulting from reduced numbers of endplate fetal AChRs, provides support for this hypothesis. The functional significance of the two AChR subtypes is not fully understood. It is thought that the longer endplate currents of fetal receptors permit newly forming neuromuscular junctions to elicit muscle contractions in the womb that are essential for fetal development (27,28). Our results demonstrate that expression of adult AChR is not essential for survival and suggest that up-regulation of AChR {gamma}-subunit expression is potentially a beneficial therapeutic strategy for AChR deficiency syndrome. A related strategy may be appropriate for other ion-channel disorders. For instance, an analogous situation occurs with glycine receptors (GlyR). Mice homozygous for the recessive 7 bp deletion (oscillator mutation) in the GlyR-{alpha}1 subunit die within 3 weeks of birth (29), whereas the loss of the GlyR-{alpha}1 subunit is partially compensated in man, leading to the hyperekplexia or ‘startle disease’ phenotype. These examples of ion-channel disorders illustrate how differences in control of gene expression may affect animal models of human neurological disorders. The transgenic mice we have generated demonstrate that more accurate models of neurological syndromes can be generated. They will be helpful for the study of present and future treatments for AChR deficiency syndrome and other myasthenic disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of mutant mice
cDNA encoding the human muscle AChR {gamma}-subunit (5) was subcloned into a plasmid vector harbouring the 2.2 kb human skeletal {alpha}-actin promoter and regulatory regions (13) and the SV40 large T poly(A) site. The {gamma}-subunit cDNA was inserted near the beginning of the second human skeletal {alpha}-actin 5' untranslated exons (21). Transgenic mice were generated by microinjection of the purified {gamma}-subunit expression cassette into the pronucleus of F2 hybrid oocytes from C57BL/6xCBA/CA parents. Positive transgenic mice were identified by analysis of PCR amplicons from genomic DNA using oligonucleotide primers 5'-GAGTTAGGGCTGAGCCAGTTCTGTG-3' and 5'-GTCTGGTGAGGGCAGGTAGGGGCGTG-3' that are specific for the human AChR {gamma}-subunit gene and amplify between exons 11 and 12. Mice with targeted disruption of the AChR {varepsilon}-subunit gene were kindly provided by Professor J. Sanes (Washington University, St Louis, USA).

Mouse breeding and genotyping
Mice harbouring the human {gamma}-subunit transgene (h{gamma}+) were crossed with mice heterozygous for the AChR {varepsilon}-subunit knock-out mutation ({varepsilon}+/–). Progeny harbouring h{gamma}+{varepsilon}+/– were mated to siblings of the same genotype to generate litters containing mice with the human {gamma}-subunit transgene in an {varepsilon}–/– background (h{gamma}+{varepsilon}–/–). Mouse genotyping was performed by PCR analysis of genomic DNA. The presence of the neomycin resistance gene, indicative of a mouse allele with the targeted disruption of the mouse {varepsilon}-subunit gene (5), was detected using primers 5'-CAACAGACAATCGGCTGCTCT-3' and 5'-GAATGGGCAGGTAGCCGGAT-3'. The mouse {varepsilon}-subunit gene was detected using primers that amplify between exons 2 and 4, 5'-CTCTTCGACAATTATGATCCAGA-3' and 5'-GAGCCGATAGTCGTGCCAGT-3'. Primers for detection of the human AChR {gamma}-subunit cDNA are described earlier.

Molecular analysis of human AChR {gamma}-subunit expression
Human AChR {gamma}-subunit mRNA expression in mouse muscle was detected by RT–PCR from total RNA extracted from limb muscle using RNA-Bee (TEL-TEST Inc., Friendswood, TX, USA). First-strand cDNA was generated using random primers and products for exons 5–8, 7–8 and 11–12, amplified using human AChR {gamma}-subunit-specific oligonucleotide primers 5'-CTTCGAGGTGGCCCTCTACTGCAA-3' and 5'-AGCACGTTGATGGCGACGGTA-3', 5'-ACCCTGAGGCCTTCACAG-3' and 5'-AGCACGTTGATGGCGACGGTA-3', and 5'-GAGTTAGGGCTGAGCCAGTTCTGTG-3' to 5'-GTCTGGTGAGGGCAGGTAGGGGCGTG-3', respectively. Mouse AChR {alpha}-subunit cDNA was detected using the primer pair 5'-AGTCCAATAACGCCGCTGAG-3' and 5'-TTTCCTAGCGATGGCTATGG-3'. Northern blots were performed according to standard procedures using 7 µg of muscle total RNA and probed with a human AChR {gamma}-subunit cDNA fragment of 480 bp spanning exons 5–8, or a mouse AChR {alpha}-subunit cDNA fragment of 530 bp spanning exons 2–6 (generated using primers 5'-TGTAGTCCGGCCAGTGGAGGACCA-3' and 5'-GGCAGCAGGAGTAGAACACCCAGT-3'). Primers for mouse GAPDH were 5'-TCAACGGCACAGTCAAGGCCGAGA-3' and 5'-ATGACCTTGCCCACAGCCTTGGCAGC-3'.

Immunoprecipitation of AChR
Freshly dissected mouse leg muscle was homogenized in 10 vol. of 10 mM Tris–HCl, pH 7.4 containing protease inhibitor cocktail (P8340 Sigma–Aldrich) (5 µl/100 mg tissue). The sample was centrifuged at 16 000g for 15 min. The supernatant was discarded and the pellet was solubilized by shaking in 10 mM Tris–HCl, pH 7.4 containing 2% Triton X-100 and protease inhibitor cocktail at 4°C overnight. The extract was centrifuged for 30 min at 100 000g. The supernatant containing crude AChR extract was used for immunoprecipitation. AChR extract was incubated with 125I-{alpha}-BuTx (Amersham Biosciences) and primary mouse anti-human AChR {gamma}-subunit monoclonal antibody, mab C9 at 4°C overnight with gentle shaking. To assess the background binding, identical reactions were performed in the presence of 1000-fold excess cold {alpha}-BuTx. Carrier normal mouse serum, followed by secondary sheep anti-mouse serum, were added and incubated at room temperature for 2 h. The samples were centrifuged for 2 min, the pellets washed twice with 10 mM Tris–HCl, pH 7.4, and then counted in a gamma counter.

Quantification of endplate AChR
Quantification of AChR was based on the method described (14). Briefly, one hemi-diaphragm from each mouse was trimmed and pinned out on Sylgard 184 (Dow Corning), and incubated for 2 h at room temperature in Kreb's buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4·7H2O, 1.2 mM KH2PO4, 24.9 mM NaHCO3, 11.1 mM D-glucose and 2.5 mM CaCl2) containing 50 nM125I-{alpha}-BuTx. The hemi-diaphragm was rinsed 3x5 min with Kreb's buffer and then washed overnight at 4°C with Kreb's buffer. The tissue was fixed in 2.5% glutaraldehyde in 80% Kreb's buffer for 2 h at room temperature and then washed 3x5 min with H2O. The diaphragms were stained for acetylcholinesterase and the region containing endplates were excised and counted in a gamma counter and weighed. Two equivalent lengths of muscle devoid of endplates were also counted and weighed. The endplate-specific 125I-{alpha}-BuTx binding was expressed as cpm/mg/length of muscle fibres, allowing comparisons between different diaphragms (14).

Evaluation of muscle strength
Muscle strength was evaluated using an inverted screen test and a forelimb weight lift test essentially as described in the literature (30). For the inverted screen test, a 50 cm2 screen of wire mesh consisting of 12 mm2 of 1 mm diameter wire surrounded by a 4 cm deep wooden frame was used. The mouse was placed in the centre of the wire mesh screen and the stopwatch started. Immediately, the screen was rotated to the inverted position over 2 s, with the mouse's head declining first. The screen was held steadily 50 cm above a padded surface for 2 min. The time at which the mouse fell-off was noted with an endpoint of 2 min. Each mouse was given a score as follows: 1, 0–10 s; 2, 11–25 s; 3, 26–60 s; 4, 61–100 s; 5, 101–119 s and 6, 120 s.

For the forelimb weight lift test, the weight consisted of a ball of tangled fine gauge stainless steel wire attached to an increasing number of chain links. At the start of the experiment one link was used. A mouse was held by the middle of the tail and lowered to grasp the first weight on the laboratory bench. As it grasped the wire, it was raised again until the link was clear of the bench, at which point the stopwatch was started. A hold of 3 s was the criterion; if this was achieved the mouse was tested on the next heavier weight, after all its littermates had tried that weight. If the weight was dropped in <3 s, the time was noted, the mouse rested for about 10 s and tried once again. If it failed again, that terminated the trial for that mouse. A final total score was calculated as the product of the number of links in the heaviest chain held for the full 3 s, multiplied by the time (s) it was held. If the heaviest weight was dropped before 3 s, an appropriate intermediate value was calculated. Thus a mouse that held a 5-link weight for 3 s, but was unable to lift a 6-link weight, was assigned a score of 15 (5x3). If it held the 6-link weight for 1 second, it scored 16 [(5x3)+1].

Immunocytochemistry
Muscle tissue from freshly killed mice was frozen in isopentane cooled in liquid N2 for 15 s. Legs were sectioned in a transverse plain using a Leica CM1900 cryostat at –18°C and at a thickness of 10–15 µm and mounted on slides coated with 3-aminopropyltriethoxysilane. Sections were permeabilized in 0.1% Triton X-100 for 10 min at room temperature. Sections were then blocked with 3% BSA in PBS for 15 min at room temperature and incubated in primary antibody for 1–2 h.

Primary antibodies were: rabbit polyclonal anti-AChR {alpha}-subunit antisera used at 1 : 1000 dilution (31), and an anti-human AChR {gamma}-subunit monoclonal antibody (C9) used at 1 : 100 (32). Sections were rinsed in PBS for 10–15 min (2 changes) and incubated in fluorescent secondary antibody (anti-rabbit Alexa Fluor 568 (Molecular Probes) at 1 : 300), anti-mouse FITC (DakoCytomation) at 1 : 300, and anti- rabbit FITC (Jackson) at 1 : 200 for 1 h at room temperature in blocking buffer. Finally, sections were rinsed in PBS for between 30 min and 1 h (2–3 changes) and then fixed if required in 3% paraformaldehyde. Endplate AChR were also directly labelled using tetramethylrhodamine-labelled {alpha}-BuTx at 1 : 1000. Fluorescent images were captured using an Olympus BX60 microscope and Improvision Openlab 3.1.5 software.

Electrophysiology
Phrenic nerve/hemi-diaphragms were prepared essentially as described in the literature (33). Hemi-diaphragms were dissected and pinned out in a Sylgard-coated Petri dish containing bubbled Kreb's solution. The phrenic nerve was stimulated via a suction electrode coupled to a pulse generator (GRASS instruments S48 solid-state square wave stimulator, Quincy, USA) with an associated stimulus isolation unit. To enable measurement of evoked potentials, muscle action potential and contraction was blocked with 2.5 µM µ-conotoxin GIIIB (Peptide Institute, Japan). Recordings were made at room temperature (20–22°C) via an Axoclamp-2A amplifier (Axon Instruments, Union City, CA, USA). Nerve-evoked EPPs and spontaneous MEPPs were recorded intracellularly with conventional borosilicate glass electrodes filled with 3 M KCl (10–15 M{Omega} resistance; Harvard Apparatus, Edenbridge, Kent, UK) and filtered at 1 kHz. Impalement adjacent to an endplate was indicated by fast EPP rise time of <3 ms.

To evoke an EPP the nerve was stimulated supramaximally with platinum wire electrodes. Upon impalement, a 30 s period of equilibration was allowed before MEPP recordings were begun. If the membrane potential depolarized by >5 mV or depolarized <–55 mV the recording was abandoned for that endplate. Data signals were passed through a Humbug 50 Hz noise eliminator (Quest Scientific via Digitimer, Welwyn Garden City, UK) to reduced electrical noise on recordings. MEPPs were auto-detected with Winwcp software (Whole cell program, Strathclyde University, UK). Auto-detection was set such that all MEPPs with amplitude greater than the magnitude of the residual electrical noise were detected. Up to 30 MEPPs followed by up to 30 EPPs (stimulated at 1 Hz) were recorded per endplate for later offline analysis. Recordings from many endplates were accumulated over a 1 h period.

All MEPPs and EPPs were standardized to a resting membrane potential of –80 mV (34). Mean quantal content (m) was calculated by the direct method using the formula:

where AMPEPP is the corrected amplitude of the EPP and AMPMEPP is the amplitude of the MEPP. Mean EPPs were corrected for non-linear summation using the formula:

where E is the resting membrane potential (–80 mV) and 0.8 is the correction factor for mouse endplates (35). Values are expressed as mean±SEM. Statistical analysis was performed with InStat software (GraphPad Software, Inc., San Diego, CA, USA). A P-value of >0.05 was considered to demonstrate the lack of a significant difference between groups.


   ACKNOWLEDGEMENTS
 
The authors thank Professor Josh Sanes for providing AChR {varepsilon}-subunit knock-out mice, Dr Robert Deacon for helpful advice in assessing the transgenic mice and Dr Leslie Jacobson and Martin Brydson for technical assistance. This work is supported by the Medical Research Council UK and the Myasthenia Gravis Association/Muscular Dystrophy Campaign.


   FOOTNOTES
 
* To whom correspondence should be addressed. Email: dbeeson{at}hammer.imm.ox.ac.uk


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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