Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration.

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the "pulse- labeled" glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.

Lithium reduces the number of acetylcholine receptors in skeletal muscle.

Extended treatment of rats with lithium inhibits the increase in the number of extrajunctional acetylcholine receptors that occurs in their denervated skeletal muscle. In normal muscle, lithium reduces the number of acetylcholine receptors at neuromuscular junctions. These changes appear to be a relatively specific effect of lithium on the turnover of receptors. Skeletal muscle provides an accessible system for analyzing the role of lithium (and other cations) in the regulation of cell surface receptors. This regulation may play a role in the mechanism by which lithium prevents recurrent manic-depressive episodes.

Motor nerve sprouting and acetylcholine receptors.

Sprouting of motor nerve terminals was evoked by functional denervation of skeletal muscles brought about by presynaptic blockade or disuse. The amount of sprouting, determined by morphometric measurement, was correlated with the level of extrajunctional acetylcholine receptors. Sprouting was inhibited by blockade of acetylcholine receptors with alpha-bungarotoxin. Extrajunctional acetylcholine receptors may play an important role in eliciting motor nerve terminal sprouting.

Myasthenic immunoglobulin accelerates acetylcholine receptor degradation.

Degradation of acetylcholine receptors by cultured rat skeletal muscle cells was determined from the release of 125I from bound 125I-labeled alpha-bungarotoxin. Addition of immunoglobulin from patients with myasthenia gravis to the culture medium accelerated the degradation rate to a mean of 8.51 +/- 0.44 percent per hour, compared with the mean control rate of 3.97 +/- 0.14 percent per hour (P less than .001). A similar mechanism may possibly be involved in the autoimmune pathogenesis of myasthenia gravis in man.

Trophic regulation of acetylcholine sensitivity of muscle: effect of electrical stimulation.

Denervation of skeletal muscle results in a spread of acetylcholine sensitivity over the entire surface membrane. Electrical stimulation, programmed to mimic the normal activity pattern, was applied continuously to the denervated rat diaphragm in vivo. After 4 days, the acetylcholine sensitivity was far less in the stimulated diaphragms than in denervated controls. Muscle activity may account for "neurotrophic" regulation of the acetylcholine sensitivity.

Thymic muscle cells bear acetylcholine receptors: possible relation to myasthenia gravis.

Culture of dissociated thymus from rats and humans yielded cells identical to skeletal muscle with respect to morphology, contractility, electrophysiological properties, and the presence of acetylcholine receptors. These cells, strategically located in the thymus, may play a role in initiation of the autoimmune response against acetylcholine receptors, which is characteristic of myasthenia gravis.

Induction of suppressor cells specific for AChR in experimental autoimmune myasthenia gravis.

Suppressor cells specific for acetylcholine receptor (AChR) were induced in a population of lymphocytes previously sensitized to AChR, obtained from rats with experimental autoimmune myasthenia gravis (EAMG). The lymphocytes were cultured with the immunosuppressive drug cyclosporin A plus purified AChR for 7 days. These cells, when mixed with lymphocytes from rats with EAMG in vitro, strongly suppressed the antibody response to AChR. They did not inhibit antibody responses to an unrelated antigen, an indication that suppression was specific for AChR. This approach should be a useful way to induce specific suppressor cells from sensitized populations of lymphocytes and may be applicable in the treatment of autoimmune diseases such as myasthenia gravis.

Rapid degradation of "new" acetylcholine receptors at neuromuscular junctions.

Acetylcholine receptors at innervated neuromuscular junctions are very stable, with half-lives reported to be 6 to 13 days. Their turnover is described as a first-order process, implying a single population of receptors. In this study, two subpopulations of acetylcholine receptors at normally innervated junctions have been identified. One has a rapid turnover rate with a half-life of 18.7 hours, similar to that of extrajunctional receptors, and the other has a slow turnover rate with a half-life of 12.4 days. The rapidly turned over subpopulation represents approximately 20 percent of the total junctional receptors. This finding may account for the discrepancies in previous reports of turnover rates and may explain the rapid reversibility in vivo of agents that "irreversibly" block acetylcholine receptors. This finding also implies that the synthesis rate of junctional acetylcholine receptors may be higher than previous estimates. The rapidly turned-over subpopulation may represent receptors that were newly inserted into the neuromuscular junction and that were not yet stabilized by an influence of the motor nerve.

Effect of myasthenic immunoglobulin on acetylcholine receptors of intact mammalian neuromuscular junctions.

Degradation of acetylcholine receptors of intact mouse neuromuscular junctions was determined in vivo and in vitro by the release of radioactivity from mouse diaphragms labeled with 125I-alpha-bungarotoxin. Treatment of mice with immunoglobulin from myasthenic patients accelerated the degradation rate to approximately three times normal, in both intact animals and organ cultures. The released radioactivity was in the form of [125I]tyrosine, confirming the nature of the degradative process. Accelerated degradation of acetylcholine receptors at neuromuscular junctions may represent an important antibody-mediated mechanisms in myasthenia gravis.

Alcoholic rhabdomyolysis: an experimental model in the rat.

The main features of alcoholic rhabdomyolysis-skeletal muscle necrosis, marked elevation of serum creatine phosphokinase, and myoglobinuria-were produced in rats by a combination of relatively prolonged (2 to 4 weeks) exposure to ethanol and a brief period of food deprivation. This observation suggests that fasting may similarly trigger muscle injury during binge drinking in man. The effect of fasting is in part related to an increase in blood alcohol due to reduced alcohol clearance and in part caused by a fasting-induced potentiation of the toxic effects of high concentrations of alcohol of skeletal muscle.

Botulinum toxin: mechanism of presynaptic blockade.

The mechanism of action of botulinum toxin was analyzed by the use of calcium ionophores and black widow spider venom. Addition of calcium ionophores to nerve-muscle preparations blocked by botulinum toxin did not increase the frequency of miniature end plate potentials. However, the spider venom elicited a barrage of miniature end plate potentials after blockade by botulinum. Electron micrographs of preparations treated with botulinum toxin and then the spider venom revealed clumping of synaptic vesicles at release sites in the otherwise depleted nerve terminals. These findings indicate that the action of botulinum toxin is not due to deficient storage of acetylcholine in vesicles or blockade of calcium entry into nerve terminals. They suggest that the toxin interferes with the acetylcholine release process itself, possibly by blocking exocytosis at the release sites.

Blockade of acetylcholine receptors: a model of myasthenia gravis.

In order to block acetylcholine receptors of muscle, the alpha toxin of the Formosan cobra (Naja naja atra) was given intravenously to rats. Electrophysiological and pharmacological changes typical of myasthenia gravis were recorded, including decremental responses to repetitive stimuli, curare sensitivity, neostigmine reversal, and posttetanic phenomena. This model supports the concept that a reduction of available acetylcholine receptors may play an important role in myasthenia gravis.

Neuromuscular junction in myasthenia gravis: decreased acetylcholine receptors.

The number of acetylcholine receptors was determined in the neuromuscular junctions of eight patients with typical myasthenia gravis and in five controls, by means of (125)1-labeled alpha-bungarotoxin binding. The junctional acetylcholine receptors were reduced in the myasthenic muscles as compared with the controls. This reduction in receptors may account for the defect in neuromuscular transmission in myasthenia gravis.

Susceptibility of skeletal muscle to Coxsackie A2 virus infection: effects of botulinum toxin and denervation.

Coxsackie A viruses can infect denervated but not innervated mature skeletal muscles. The role of synaptic transmission in preventing susceptibility to Coxsackievirus infection was studied by surgically denervating leg muscles of mice or injecting the muscles with botulinum toxin to block quantal release of acetylcholine. Control muscles were injected with heat-inactivated toxin. Subsequent injection of Coxsackie A2 virus resulted in extensive virus replication and tissue destruction in the denervated and botulinum toxin-treated muscles, while the control muscles showed only minimal changes. This suggests that the susceptibility of skeletal muscle to Coxsackievirus infection is regulated by synaptic transmission.

Granzyme B: evidence for a role in the origin of myasthenia gravis.

PURPOSE OF RESEARCH: Although the pathogenesis of myasthenia gravis (MG) as an antibody mediated disorder of acetylcholine receptors (AChRs) at neuromuscular junctions is well understood, the origin of the autoimmune response is unclear. The thymus is intimately involved in initiation of the autoimmune response; the antigen, AChR, is present in the thymus, but how the autoimmune response is triggered is not known. Granzyme B (GrB), a proteolytic enzyme present in cytolytic T cells and natural killer (NK) cells, selectively cleaves many potential autoantigens (but few non-autoantigens), generating novel fragments that trigger autoreactive responses. This protease has been strongly implicated in the pathogenesis of several autoimmune diseases including lupus, rheumatoid arthritis, dermatomyositis, and others. In the studies described in this manuscript, we examined the ability of GrB to cleave the AChR subunits, and performed biochemical, immunohistochemical and molecular studies on thymus glands from myasthenic patients and controls to assess GrB expression. MAIN RESULTS: GrB efficiently and specifically cleaves subunits of AChR, especially the epsilon subunit. GrB is present in thymus glands from myasthenia patients, but is absent in control thymuses. CONCLUSIONS: Our results provide evidence supporting a potential role for GrB in the process of initiation of MG, and are consistent with the concept of an immunodominant epsilon epitope.

Molecular mimicry and myasthenia gravis. An autoantigenic site of the acetylcholine receptor alpha-subunit that has biologic activity and reacts immunochemically with herpes simplex virus.

The large majority of patients with the autoimmune disease myasthenia gravis characteristically have detectable antibodies against the acetylcholine receptor (AChR). We used synthetic peptides to identify antibodies in sera of myasthenia gravis patients reactive with the human acetylcholine receptor (HuAChR) alpha-subunit, residues 160-167. Affinity purification of these antibodies, using the HuAChR alpha-subunit 157-170 peptide immobilized on thiopropyl-Sepharose, yielded IgG antibodies that bound to the native AChR and inhibited the binding of alpha-bungarotoxin to the receptor. The HuAChR alpha-subunit 160-167 peptide demonstrated specific immunological cross-reactivity with a shared homologous domain on herpes simplex virus glycoprotein D, residues 286-293, by both binding and inhibition studies. Thus, HuAChR alpha-subunit, residues 160-167, elicits antibodies in myasthenic patients that binds to the native AChR protein and is capable of eliciting a biologic effect. Immunologic cross-reactivity of this "self" epitope with herpes simplex virus suggest that this virus may be associated with the initiation of some cases of myasthenia.

The pathogenesis of reactive axonal swellings: role of axonal transport.

The role of axonal transport in the pathogenesis of the axonal swellings which develop at the severed ends of transected axons was studied by electron microscopic (EM) autoradiography. Proteins carried by fast anterograde transport in rat sciatic nerves were labeled with [3H]-leucine or [3H]-fucose; [3H]-leucine, [3H]-fucose, and [125I]-tetanus toxin were used to label components of retrograde transport. After the labeling procedure, the nerves were ligated and 2 to 24 hours later the animals were perfused with fixatives. The axonal swellings in both the proximal and distal stumps contained densely packed membranous organelles. The transported radioactivity in the swellings was strictly associated with these organelles, particularly pleomorphic vesicles and branched tubules derived from smooth endoplasmic reticulum. The endogenous (tritiated) substances had a similar association with the organelle collections in both the proximal stump (fast anterograde transport) and in the distal stump (retrograde transport). The exogenous marker of retrograde transport (125I-tetanus toxin) had the same autoradiographic localization. These results suggest that fast anterograde and retrograde transport are very similar processes carrying predominantly membranous organelles and constituting a system of bidirectional fast transport. The accumulations of organelles in reactive swellings are interpreted as the consequence of the acute focal interruption of this system. Studies of axonal transport provide a means for investigation of the origin and fate of axonal organelles in pathologic processes.

Are muscle fibers denervated in myotonic dystrophy?

An underlying neurogenic abnormality has recently been postulated in the muscular dystrophies. To test this hypothesis, we applied a widely accepted criterion of denervation-ie, and increase in extrajunctional acetyicholine (ACh) receptor sites--to muscles biopsy specimens from nine patients with myotonic dystrophy and three with amyotrophic lateral scierosis (ALS). The ACh receptor sites were determined by means of iodine 125-labeled alpha-bungarotoxin binding, measured by scintillation counting and autoradiography. None of the myotonic dystrophy muscles showed increased extrajunctiona ACh receptor sites, even in the smallest fibers. By contrast, muscle biopsy specimens from patients with ALS showed notably increased extrajunctional ACh receptor sites, especially in the small fibers. Our findings do not support the hypothesis of a neurogenic defect in myotonic dystrophy.

Prednisone treatment in Duchenne muscular dystrophy. Long-term benefit.

We report the long-term results of a therapeutic trial of prednisone in the treatment of Duchenne muscular dystrophy, comparing the age at which 16 treated patients and 38 controls lost the ability to ambulate. Survival curve analysis and group mean comparisons indicate that ambulation was significantly prolonged, by approximately two years, in the prednisone-treated group compared with the control group. The results of this study indicate that prednisone therapy has a long-term beneficial effect in the treatment of Duchenne muscular dystrophy, in addition to the short-term palliation we previously reported. Further studies are needed to establish optimal treatment schedules using prednisone and to develop improved therapeutic agents.

Experimental arthrogryposis caused by viral myopathy.

Immobilization of the embryo has been postulated to cause the joint deformities in arthrogryposis multiplex congenita (AMC). Experimental damage to the motor neurons or pharmacologic blockade of neuromuscular transmission has previously resulted in typical joint changes of AMC. In the present investigation, we have studied the effects of paralysis produced by a viral myopathy on joint development. Coxsackievirus A2 was injected intravenously into chick embryos on the seventh day of incubation. Within 48 hours, severe myositis and paralysis resulted. Electron microscopical and immunofluorescence techniques demonstrated virus in muscle cells. Within three to four days after infection, the muscle had virtually disappeared. Ankylosis of joints, corresponding to that seen in human AMC, occurred. This study shows that primary myopathy with paralysis can produce arthrogrypotic joint deformities. The possibility of a viral etiologic factor in some human cases of AMC should be considered.