Orna Asher

Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor.

Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR α subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR α subunit (Hα1 – 210), and to portions of it that encompass either the MIR (Hα1 – 121) or the ligand binding site of AChR (Hα122 – 210), and studied their ability to interfere with the immunopathological anti‐AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S‐transferase. Fragments Hα1 – 121 and Hα1 – 210 protected AChR in TE671 cells against accelerated degradation induced by the anti‐MIR monoclonal antibody (mAb)198 in a dose‐dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti‐MIR mAb and by polyclonal rat anti‐AChR antibodies. Fragments Hα1 – 121 and Hα1 – 210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment Hα122 – 210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen‐specific therapy of myasthenia.

Acetylcholine receptor gene expression in experimental autoimmune myasthenia gravis.

Acetylcholine receptor (AChR) gene expression was analyzed in experimental autoimmune myasthenia gravis (EAMG) in rabbits, rats and mice. An increase in AChR transcripts was demonstrated to be exclusively associated with myasthénie symptoms and with a severe loss in membrane AChR. An increase of α‐, β‐, ϵ‐, and δ‐subunit specific mRNAs (5.2‐, 1.6‐, 3.2‐ and 3.7‐fold, respectively), which code for the adult type of AChR (α2βϵδ) was observed in EAMG in rats. The γ‐subunit transcript was not detectable in myasthenie or healthy rats. It appears that the regulatory control of AChR gene expression in EAMG is different from that observed upon denervation.

Increased levels of acetylcholine receptor α‐subunit mRNA in experimental autoimmune myasthenia gravis

To gain insight into the regulatory mechanisms underlying the blockade and loss of acetylcholine receptor (AChR) in myasthenia, we have followed AChR α‐subunit mRNA levels in leg muscles of myasthenic and normal rabbits and rats. Northern blots of RNA preparations from normal and myasthenic animals were hybridized with a mouse AChR α‐subunit cDNA probe. Our experiments indicate a specific increase (4–7‐fold) in the levels of α‐subunit mRNA in animals with experimental autoimmune myasthenia gravis (EAMG), in comparison with control animals. Actin mRNA levels were essentially unchanged. Our results thus suggest that EAMG is accompanied by an increased level of AChR gene transcription.

Regulation of acetylcholine receptor gene expression in rats treated with α‐bungarotoxin

Regulation of acetylcholine receptor (AChR) gene expression was analyzed in α‐bungarotoxin (α‐BTX) treated rats. A reduction in available 125l‐α‐BTX binding sites was accompanied by an increase in the various AChR transcripts. The increase in the AChR α‐, β‐ c‐ and δ‐subunit mRNAs was similar to that observed in rats with experimental autoimmune myasthenia gravis (EAMG). Unlike in EAMG, the γ‐subunit transcripts reappeared following α‐BTX treatment. The quantitative differences in the levels of AChR transcripts between α‐BTX treatment and EAMG on one hand and denervation on the other hand, support the notion that the regulation of AChR gene expression is controlled by muscle activity and by neuronal factors as well. We also demonstrate in this report that myogenin transcripts increase following α‐BTX treatment as well as following denervation, whereas MyoD1 transcripts remain stable.

Changes in the expression of mRNAs for myogenic factors and other muscle-specific proteins in experimental autoimmune myasthenia gravis

The regulation of genes for acetylcholine receptor (AChR), myogenic factors and other muscle‐specific proteins has been analyzed in experimental autoimmune myasthenia gravis (EAMG) and following denervation. The levels of the transcripts for the myogenic factors, MyoDl, myogenin and MRF4, were measured using Northern blot analysis. Myogenin and MRF4 transcript levels were observed to be 3.1 ‐ and 2.6‐fold higher in muscle of rats with EAMG than in controls, respectively. MyoDl levels, however, remained unchanged. The increases in AChR, myogenin and MRF4 mRNAs were one order of magnitude higher in 2‐week denervated muscle than in the myasthenic muscle. The levels of muscle creatine kinase (MCK), α‐actin and muscle dystrophin transcripts were also analyzed. Dystrophin levels were found to be 1.7‐ and 4.7‐fold higher in EAMG and denervated muscle, respectively, than in controls; in contrast, MCK and α‐actin levels remained unchanged.

Functional characterization of mongoose nicotinic acetylcholine receptor α-subunit: resistance to α-bungarotoxin and high sensitivity to acetylcholine

The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) α‐subunit contains a number of mutations in the ligand‐binding domain and exhibits poor binding of α‐bungarotoxin (α‐BTX). We characterized the functional properties of a hybrid (α‐mongoose/βγδ‐rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of α‐BTX to the hybrid AChR was 200‐fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 μM for the rat AChR). Hence, changes in the ligand‐binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

The mongoose acetylcholine receptor α-subunit: analysis of glycosylation and α-bungarotoxin binding

The mongoose AChR α‐subunit has been cloned and shown to be highly homologous to other AChR α‐subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind α‐bungarotoxin (α‐BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn‐187, forms a consensus N‐glycosylation site. The mongoose glycosylated α‐subunit has a higher apparent molecular mass than that of the rat glycosylated α‐subunit, probably resulting from the additional glycosylation at Asn‐187 of the mongoose subunit. The in vitro translated mongoose α‐subunit, in a glycosylated or non‐glycosylated form, does not bind α‐BTX, indicating that lack of α‐BTX binding can be achieved also in the absence of glycosylation.

Acetylcholine receptor and myogenic factor gene expression in Torpedo embryonic development.

The mRNA levels of acetylcholine receptor (AChR) and myogenic factors were followed during embryonic development of Torpedo skeletal muscle and its homologue, the electric organ. A different developmental pattern of AChR gene expression was found in these two tissues: a slight decrease in the muscle, and a marked increase, concomitant with synapse formation, in the electric organ. However, the developmental pattern of MyoD and MRF4 mRNA levels was similar in both tissues, with no significant changes during development. This is in contrast with the sharp increase in the expression of AChR in the electric organ and may suggest that the burst in the expression of AChR during the differentiation of myotubes into electrocytes is not regulated by changes in the myogenic factor mRNA levels.

Evidence for an Upregulation of Acetylcholine Receptor Messenger Subunits Triggered by Antibody-Mediated Receptor Internalization in Human Myasthenia Gravis Muscles

The muscle nicotinic acetylcholine receptor (nAChR) is a well-characterized, ligand-gated ion channel of 270 kD that forms a pentameric complex of four homologous subunits with a molar stoichiometry of α2, β, e, δ [1, 2]. In mammalian muscle, regulation of nAChR subunit mRNAs and the distribution of nAChRs along the fiber are developmentally regulated. Transcript levels increase during myogenic differentiation and are repressed during muscle innervation; denervation results in re-accumulation [3]. The nAChR e and γ-subunits are also under developmental control, with expression of two types of channels in mammals. In early embryonic stages and upon denervation, a low-conductance, long-open-time channel composed of α, β, δ and γ-subunits and distributed throughout the fiber length predominates [4], whereas in adult fibers, a high-conductance, brief-open-time channel containing α, β, δ and e-subunits is expressed exclusively at the motor-endplate [5].