Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance.

BACKGROUND AND PURPOSE: The cholinergic agonist levamisole is widely used to treat parasitic nematode infestations. This anthelmintic drug paralyses worms by activating a class of levamisole-sensitive acetylcholine receptors (L-AChRs) expressed in nematode muscle cells. However, levamisole efficacy has been compromised by the emergence of drug-resistant parasites, especially in gastrointestinal nematodes such as Haemonchus contortus. We report here the first functional reconstitution and pharmacological characterization of H. contortus L-AChRs in a heterologous expression system. EXPERIMENTAL APPROACH: In the free-living nematode Caenorhabditis elegans, five AChR subunit and three ancillary protein genes are necessary in vivo and in vitro to synthesize L-AChRs. We have cloned the H. contortus orthologues of these genes and expressed them in Xenopus oocytes. We reconstituted two types of H. contortus L-AChRs with distinct pharmacologies by combining different receptor subunits. KEY RESULTS: The Hco-ACR-8 subunit plays a pivotal role in selective sensitivity to levamisole. As observed with C. elegans L-AChRs, expression of H. contortus receptors requires the ancillary proteins Hco-RIC-3, Hco-UNC-50 and Hco-UNC-74. Using this experimental system, we demonstrated that a truncated Hco-UNC-63 L-AChR subunit, which was specifically detected in a levamisole-resistant H. contortus isolate, but not in levamisole-sensitive strains, hampers the normal function of L-AChRs, when co-expressed with its full-length counterpart. CONCLUSIONS AND IMPLICATIONS: We provide the first functional evidence for a putative molecular mechanism involved in levamisole resistance in any parasitic nematode. This expression system will provide a means to analyse molecular polymorphisms associated with drug resistance at the electrophysiological level.

Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line.

Transposons have been enormously useful for genetic analysis in both Drosophila and bacteria. Mutagenic insertions constitute molecular tags that are used to rapidly clone the mutated gene. Such techniques would be especially advantageous in the nematode Caenorhabditis elegans, as the entire sequence of the genome has been determined. Several different types of endogenous transposons are present in C. elegans, and these can be mobilized in mutator strains (reviewed in ref. 1). Unfortunately, use of these native transposons for regulated transposition in C. elegans is limited. First, all strains contain multiple copies of these transposons and thus new insertions do not provide unique tags. Second, mutator strains tend to activate the transposition of several classes of transposons, so that the type of transposon associated with a particular mutation is not known. Here we demonstrate that the Drosophila mariner element Mos1 can be mobilized in C. elegans. First, efficient mobilization of Mos1 is possible in somatic cells. Second, heritable insertions of the transposon can be generated in the germ line. Third, genes that have been mutated by insertion can be rapidly identified using inverse polymerase chain reaction. Fourth, these insertions can subsequently be remobilized to generate deletion and frameshift mutations by imperfect excision.

Role of the target organ in determining susceptibility to experimental autoimmune myasthenia gravis.

Injection of anti-AChR antibodies in passive transfer experimental autoimmune myasthenia gravis (EAMG) results in increased degradation of acetylcholine receptor (AChR) and increased synthesis of AChR alpha-subunit mRNA. Passive transfer of anti-Main Immunogenic Region (MIR) mAb 35 in aged rats does not induce clinical signs of disease nor AChR loss. The expression of the AChR subunit genes was analyzed in susceptible and resistant rats. In aged EAMG resistant rats, no increase in the amount of AChR alpha-subunit mRNA was measured. In vivo AChR degradation experiments did not show any increase in AChR degradation rates in aged resistant rats, in contrast to young susceptible rats. Taken together, these data demonstrate that resistance of the AChR protein to antibody-mediated degradation is the primary mechanism that accounts for the resistance to passive transfer EAMG in aged rats.

Nonmyogenic factors bind nicotinic acetylcholine receptor promoter elements required for response to denervation.

Nicotinic acetylcholine receptors (AChRs) belong to a class of muscle proteins whose expression is regulated by muscle electrical activity. In innervated muscle fiber, AChR genes are transcriptionally repressed outside of the synapse, while after denervation they become reexpressed throughout the fiber. The myogenic determination factors (MDFs) of the MyoD family have been shown to play a central role in this innervation-dependent regulation. In the chicken AChR alpha-subunit gene promoter, two E-boxes that bind MDFs are necessary to achieve the enhancement of transcription following muscle denervation. However, the deletion of promoter sequences located upstream to these E-boxes greatly impairs the response to denervation (Bessereau, J. L., Stratford- Perricaudet, L. D., Piette, J., Le Poupon, C. and Changeux, J. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1304-1308). Here we identified two additional cis-regulatory elements of the alpha-subunit gene promoter that cooperate with the E-boxes in the denervation response. One region binds the Sp1 and Sp3 zinc finger transcription factors. The second region binds at least three distinct factors, among which we identified an upstream stimulatory factor, a b-ZIP-HLH transcription factor. We propose that among MDF-responsive muscle promoters, a specific combination between myogenic and nonmyogenic factors specify innervation-dependent versus innervation-independent promoters.

Expression and immunogenicity in rats of recombinant adenovirus 5 DNA plasmids and vaccinia virus containing the HTLV-I env gene.

The complete human T-cell leukemia virus type I (HTLV-I) env gene was inserted into an expression cassette containing the adenovirus 5 major late promoter (Ad5-MLP). Recombinant Ad5-HTLV-I-env was obtained by homologous recombination in 293 cells simultaneously transfected by the expression cassette and the genomic DNA of Ad5. In vitro expression of the HTLV-I-env gene in the recombinant vector was detected by immunofluorescence and Western blotting. Functional expression of HTLV-I-env was confirmed by syncitium formation specifically in HeLa cells infected with Ad5-HTLV-I-env. Two immunization regimens against HTLV-I were tested in WKY and Fischer F-344 rats. The first involved WKY rats primed with Ad5-HTLV-I-env or naked DNA plasmids containing the HTLV-I-env gene and boosted with Ad5 containing the HTLV-I-env gp46 gene or with baculovirus-derived recombinant gp46. No antibody against HTLV-I was detected, while HTLV-I-specific cytotoxic T lymphocytes were recovered from all immunized groups but not from controls. The second approach involved Fischer F-344 rats primed and boosted with recombinant vaccinia virus containing the HTLV-I-env gene. Such rats developed antibodies against the HTLV-I env gp21 and gp46 (non-neutralizing). After challenge with human HTLV-I-producing cells (MT-2), both immunization regimens were found to induce partial protection.

ErbB3 and ErbB2/neu mediate the effect of heregulin on acetylcholine receptor gene expression in muscle: differential expression at the endplate.

Motor neurons modulate acetylcholine receptor (AChR) gene expression in skeletal muscle by two signalling pathways: the transmitter-evoked depolarization of muscle membrane inhibits AChR gene transcription throughout the myofibre presumably via activation of a serine/threonine kinase, while the transcription rates of AChR genes in the synaptic region are increased by nerve-derived trophic factors including AChR-inducing activity (ARIA). To gain further insight into these interactions we characterized the receptor for heregulin (HRG)/ARIA in muscle. We showed that HRG increases AChR alpha-subunit mRNA levels via tyrosine phosphorylation of ErbB3 and ErbB2/neu in myotubes. The protein tyrosine phosphatase inhibitor, pervanadate, potentiated the responses to HRG that were in turn blocked by the tyrosine kinase inhibitor erstatin, indicating the relevance of tyrosine phosphorylation to these events. The effects of HRG were inhibited by enhanced cellular serine/threonine phosphorylation which has been implicated in the repression of AChR genes by electrical activity. Immunocytochemical analysis of adult rat muscle revealed that while ErbB2/neu is present throughout the entire surface of the myofibre membrane, ErbB3 expression is exclusively restricted to the endplate suggesting its involvement in synapse-specific transcription of AChR genes by HRG/ARIA.

In vivo and in vitro analysis of electrical activity-dependent expression of muscle acetylcholine receptor genes using adenovirus.

Acetylcholine receptor (AChR) genes are repressed in extrajunctional domains of adult muscle fiber by neurally evoked electrical activity. Denervation elicits upregulation of AChR gene transcription in extrasynaptic areas. We have used an adenovirus (Ad)-based strategy to analyze in vitro and in vivo the electrical activity-dependent transcription of the chicken AChR alpha 1 subunit gene. The luciferase gene placed under the control of wild-type and mutated fragments of the alpha 1 subunit promoter was inserted in a defective Ad vector designed for the study of transcriptional regulation. Animals were infected by intramuscular injection and in vivo luciferase levels were normalized by coinfection with an Ad vector containing the chloramphenicol acetyltransferase gene driven by an electrical activity-insensitive promoter. Our results demonstrate that although both proximal MyoD binding sites of the alpha 1 promoter are required for muscle-specific expression of the alpha 1 gene, only one is necessary, albeit insufficient, to enhance alpha 1 promoter activity after denervation. Parallel results were obtained with cultured muscle cells in vitro following tetrodotoxin blocking of spontaneous electrical activity. These results substantiate a direct contribution of MyoD factors in electrical activity-dependent regulation of AChR expression and further indicate that Ad-based vectors constitute a powerful tool in the field of transcriptional regulation.

Muscle-specific expression of the acetylcholine receptor alpha-subunit gene requires both positive and negative interactions between myogenic factors, Sp1 and GBF factors.

The dependence of the muscle-specific enhancer of the acetylcholine receptor alpha-subunit gene on other domains of the promoter has been analysed by performing point mutagenesis and modular reconstitution of the enhancer--promoter sequences. The enhancer is inactive in the absence of the proximal region containing an Sp1 binding site and an overlapping G-C homopolymer binding factor site (referred to as GBF). The proximal region can be replaced by an Sp1 binding site from SV40 or an MEF-2 binding site from the muscle creatine kinase gene. Specific mutation of the Sp1 site markedly affects transactivation by CMD1 or myogenin. Mutation of the GBF binding site leads to higher promoter activity in primary cultures of chick myotubes or in quail fibroblasts. In addition, binding of a purified Sp1 protein prevents the binding of GBF in vitro. It is proposed that in the case of the alpha-subunit promoter, the myogenic factors activate transcription in cooperation with Sp1, and that GBF contributes to muscle-specific expression of the promoter by interfering with Sp1 binding in nonmuscle muscle cells or myoblasts.

An acetylcholine receptor alpha-subunit promoter conferring preferential synaptic expression in muscle of transgenic mice.

We have obtained transgenic mice expressing nuclearly targeted beta-galactosidase (nls-beta-gal) under the control of a chicken acetylcholine receptor alpha-subunit promoter. The expression of the transgene was detected in early somites, starting before embryonic day 9.5. In 13-day embryos, the expression pattern of the transgene closely paralleled that of the endogenous mouse alpha-subunit gene, assessed by in situ hybridization. Our results illustrate, with single-cell resolution, the tissue specificity of this alpha-subunit promoter during embryogenesis. After birth, the overall beta-galactosidase activity rapidly decreased with age. However, in diaphragms of newborn animals, beta-galactosidase activity selectively persisted in nuclei underlying the motor endplates. The latter were revealed by an acetylcholinesterase stain. Nls-beta-gal was also visualized by indirect immunofluorescence, while endplates were labelled with fluorescent alpha-bungarotoxin. Confocal microscopy unambiguously identified the more intensely stained nuclei as synaptic 'fundamental nuclei', and allowed estimates of relative staining levels. Thus an 842 bp acetylcholine receptor gene promoter confers preferential synaptic expression to a reporter gene within myofibres in vivo.

Two adjacent MyoD1-binding sites regulate expression of the acetylcholine receptor alpha-subunit gene.

Several genes encoding putative myogenic regulatory factors have been isolated on the basis of their ability to convert nonmuscle cells into myoblasts. Four of these genes code for nuclear proteins that belong to a larger family characterized by a conserved helix-loop-helix motif required for DNA-binding and dimerization. At least one protein, MyoD1, can function as a transcription factor and activate muscle-specific genes during differentiation. But the promoter of the delta-subunit gene of the mouse acetylcholine receptor (AChR) was recently reported to be functional in the absence of MyoD1 binding sites and it has been suggested that the genes coding for the AChR could be regulated independently of MyoD1 protein. Here, we identify two functional MyoD1-binding sites in the muscle-specific enhancer of the chicken AChR alpha-subunit gene that are essential for full activity in transfected myotubes.

Denervation of mouse skeletal muscle differentially affects the expression of the jun and fos proto-oncogenes.

The mRNA levels of the jun and fos proto-oncogenes, whose products are part of the transcription regulatory complex AP1, were investigated after denervation of the lower-leg muscles of adult mice. The levels of the four transcripts studied varied in a non-coordinated manner after muscle denervation. As early as 1.5 h after sciatic nerve section, c-fos mRNA reached a maximum and then returned to basal level at 6 h, while c-jun and jun-B mRNA levels increased after 24 h and remained elevated for at least 8 days. In contrast, jun-D mRNA levels did not change after denervation. In situ hybridization experiments revealed different patterns of spatial localization of the c-jun and jun-D transcripts. Labeling of dividing cells by the thymidine analog bromo-deoxyuridine demonstrated that proliferating cells were at least 40-fold less abundant than those expressing c-jun. Thus, denervation rapidly and differentially modulates the expression of genes coding for the AP1 transcription factor subunits in differentiated muscle cells. The possibility that these proto-oncogenes might be involved in the trans-synaptic regulation of muscle-specific gene expression is discussed.