Many diseases involve disruption of chemical transmission between neurons and their target cells. By analyzing such abnormalities researchers have learned a great deal about the mechanisms underlying normal synaptic transmission as well as disorders caused by dysfunction at the synapse.
Diseases that disrupt transmission at the neuromuscular junction fall into two broad categories: those that affect the presynaptic terminal and those that primarily involve the postsynaptic membrane. In both categories the most intensively studied cases are autoimmune and inherited defects in critical synaptic proteins.
Myasthenia Gravis Is the Best Studied Example of a Neuromuscular Junction Disease
The most common and extensively studied disease affecting synaptic transmission is myasthenia gravis, a disorder at the neuromuscular junction in skeletal muscle. Myasthenia gravis (the term means severe weakness of muscle) has two major forms. The most prevalent is the autoimmune form. The second is congenital and heritable; it is not an autoimmune disorder and is heterogeneous. Fewer than 500 cases have been identified, but analysis of the congenital syndromes has provided information about the organization and function of the human neuromuscular junction. This form is discussed later in the chapter.
In autoimmune myasthenia gravis antibodies are produced against the nicotinic acetylcholine (ACh) receptor in muscle. These antibodies interfere with synaptic transmission by reducing the number of functional receptors or by impeding the interaction of ACh with its receptors. As a result, communication between the motor neuron and the skeletal muscle becomes weakened. This weakness has four special characteristics:
It almost always affects cranial muscles—eyelids, eye muscles, and oropharyngeal muscles—as well as limb muscles.
The severity of symptoms varies in the course of a single day, from day to day, or over longer periods (giving rise to periods of remission or exacerbation), making myasthenia gravis unlike most other diseases of muscle or nerve.
There are no conventional clinical or electromyographic signs of denervation.
The weakness is reversed by drugs that inhibit acetylcholinesterase, the enzyme that degrades ACh.
Myasthenia gravis is a disorder of neuromuscular transmission. When a motor nerve is stimulated at rates of 2 to 5 per second, the amplitude of the compound action potential evoked in normal human muscle remains constant. In myasthenia gravis the amplitude decreases rapidly. This abnormality resembles the pattern induced in normal muscle by d-tubocurarine (the active compound in curare), which blocks nicotinic ACh receptors and inhibits the action of ACh at the neuromuscular junction. Neostigmine (Prostigmin), which inhibits acetylcholinesterase and thus prolongs the action of ACh at the neuromuscular junction, reverses the decrease in amplitude of evoked compound action potentials in myasthenic patients (Figure 14–5).
Synaptic transmission at the neuromuscular junction fails in myasthenia gravis.
(Reproduced, with permission, from Lisak and Barchi 1982.)
A. In the normal neuromuscular junction the amplitude of the end-plate potential is so large that all fluctuations in the potential occur well above the threshold for an action potential. That is, there is a large safety factor in synaptic transmission (1). Therefore, during repetitive stimulation of the motor nerve the amplitude of the compound action potentials, representing the action potentials in all muscle fibers innervated by the nerve, is constant and invariant (2).
B. In the myasthenic neuromuscular junction postsynaptic changes reduce the amplitude of the end-plate potential so that under optimal circumstances the end-plate potential may be just sufficient to produce a muscle action potential. Fluctuations in transmitter release that normally accompany repeated stimulation now cause the end-plate potential to drop below this threshold, leading to conduction failure at that synapse (1). The amplitude of the compound action potentials in the muscle declines progressively and shows only a small and variable recovery (2).
The decrease in the compound muscle action potential in response to repetitive stimulation of the motor nerve mirrors the clinical symptom of fatigability in myasthenia. For example, when patients are asked to look upward in a sustained gaze, the eyelids tire after several seconds and droop downward (ptosis). Like decremental responses on electromyography, this fatigability and drooping reverse after treatment with inhibitors of acetylcholinesterase (Figure 14–6).
Myasthenia gravis often selectively affects the cranial muscles.
(Reproduced, with permission, from Rowland, Hoefer, and Aranow 1960.)
A. Severe drooping of the eyelids, or ptosis, is characteristic of myasthenia gravis. This patient also could not move his eyes to look to either side.
B. One minute after an intravenous injection of 10 mg edrophonium, an inhibitor of acetylcholinesterase, both eyes are open and can be moved freely. The inhibition of acetylcholinesterase prolongs the action of ACh in the synaptic cleft, thus compensating for the reduced number of ACh receptors in the muscle (see Figure 14–7).
Approximately 15% of adult patients with myasthenia have benign tumors of the thymus (thymomas). As the symptoms in myasthenic patients are often improved by removal of these tumors, some element of the thymoma may stimulate autoimmune pathology. Indeed, myasthenia gravis often affects people who have other autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, or Graves disease (hyperthyroidism).
The modern concept of myasthenia emerged with the isolation and characterization of the nicotinic ACh receptor. In 1973 Douglas Fambrough and Daniel Drachman, using radioactive α-bungarotoxin to label the receptor in human end-plates, found fewer binding sites in myasthenic muscle than in controls. In addition, morphological studies revealed a smoothing of the junctional folds, the site of receptor localization (Figure 14–7).
Morphological abnormalities of the neuromuscular junction in myasthenia gravis.
At the neuromuscular junction ACh is released by exocytosis of synaptic vesicles at active zones in the nerve terminal. Acetylcholine flows across the synaptic cleft to reach receptors that are concentrated at the peaks of junctional folds. Acetylcholinesterase in the cleft rapidly terminates transmission by hydrolyzing ACh. The myasthenic neuromuscular junction has a reduced number of ACh receptors, simplified synaptic folds, and a widened synaptic space, but a normal nerve terminal.
That same year James Patrick and Jon Lindstrom demonstrated in rabbits that the generation of antireceptor antibodies was accompanied by the onset of myasthenia-like symptoms when they injected animals with nicotinic ACh receptors purified from eel electroplax (which is related to the skeletal muscles of higher vertebrates). The weakness was reversed by the cholinesterase inhibitors neostigmine or edrophonium. As in humans with myasthenia gravis, the animals were abnormally sensitive to neuromuscular blocking agents such as curare, and the evoked compound action potentials in muscle decreased with repetitive stimulation. It was later found that a similar syndrome could be induced in mice and other mammals by immunization with nicotinic ACh receptor protein (Figure 14–8).
The posture of a myasthenic mouse improves after treatment with neostigmine.
To produce the syndrome the mouse was immunized with 15 μg of purified nicotinic ACh receptor protein. (Reproduced, with permission, from Berman and Patrick 1980.)
A. Before treatment the mouse is inactive.
B. The mouse is standing 12 minutes after receiving an intraperitoneal injection of 37.5 μg/kg neostigmine bromide, which inhibits acetylcholinesterase and thus increases the availability of ACh in the synaptic cleft of the neuromuscular junction.
By 1975 all the essential characteristics of the human disease had been reproduced in experimental autoimmune myasthenia gravis in mice, rabbits, and monkeys. After experimental myasthenia gravis was characterized, antibodies directed against nicotinic ACh receptors were found in the serum of many patients with myasthenia. How do these immunological observations account for the characteristic decrease in the response of myasthenic muscle to repeated stimulation?
An action potential in a motor axon normally releases enough ACh to induce a large excitatory end-plate potential with an amplitude of approximately 70 to 80 mV relative to the resting potential of –90 mV (see Chapter 9). Thus the normal end-plate potential is greater than the threshold needed to initiate an action potential, approximately –45 mV. In normal muscle the difference between the threshold and the actual end-plate potential amplitude—the safety factor—is therefore quite large (Figure 14–5A). In fact, in many muscles the amount of ACh released during synaptic transmission can be reduced to as little as 25% of normal before it fails to initiate an action potential.
A reduction in the density of ACh receptors, as in myasthenia, reduces the probability that a molecule of ACh will find a receptor before it is hydrolyzed by the enzyme acetylcholinesterase. In addition, in myasthenia the geometry of the end-plate is also disturbed. The normal infolding at the junctional folds is reduced and the synaptic cleft is enlarged (Figure 14–7). These morphological changes promote diffusion of ACh away from the synaptic cleft and thus further reduce the probability of ACh interacting with the few remaining functional receptors. As a result, the amplitude of the end-plate potential is reduced to the point where it is barely above threshold (Figure 14–5B).
Thus in myasthenia synaptic transmission is readily blocked even though the vesicles in the presynaptic terminals contain normal amounts of ACh and the process of transmitter release is intact. Both the physiological abnormality (the decremental response) and the clinical symptoms (muscle weakness) are partially reversed by drugs that inhibit acetylcholinesterase (Figures 14–6 and 14–8).
How do antibodies cause the symptoms of myasthenia? The antibodies do not simply occupy the site of ACh binding. Rather, they appear to react with epitopes elsewhere on the receptor molecule. This increases the destruction of nicotinic ACh receptors, probably because myasthenic antibodies bind and cross-link the receptors, triggering their degradation (Figure 14–9). In addition, some myasthenic antibodies bind proteins of the complement cascade of the immune system, causing lysis of the postsynaptic membrane.
Turnover of ACh receptors increases in myasthenia.
(Adapted, with permission, from Lindstrom 1983, and Drachman 1983.)
A. Normal turnover of randomly spaced ACh receptors takes places every 5 to 7 days.
B. In myasthenia gravis and experimental myasthenia gravis, the cross-linking of ACh receptors by antibodies facilitates endocytosis and the phagocytic destruction of the receptors, which leads to a two- to threefold increase in the rate of receptor turnover. Binding of antireceptor antibody activates the complement cascade, which is involved in focal lysis of the postsynaptic membrane. This focal lysis is probably primarily responsible for the characteristic morphological alterations of postsynaptic membranes in myasthenia (see Figure 14–7).
Despite the evidence documenting the primary role of antibodies against the nicotinic ACh receptor in myasthenia, approximately one-fifth of patients with myasthenia, including some who respond to anti-immune therapy like plasmapheresis, do not have these antibodies. Instead, most of these patients have antibodies against another postsynaptic protein, known as MuSK (muscle-specific Trk-related receptor with a kringle domain). MuSK is a receptor tyrosine kinase that interacts with agrin, a protein released from the motor nerve terminal that helps to organize the nicotinic ACh receptors into clusters at the neuromuscular junction. It appears to be functionally important both during development and in the adult. Although the adverse effects of the anti-MuSK antibodies have not yet been defined, the antibodies block some of the normal clustering of the nicotinic ACh receptors following the interaction of agrin with MuSK.
Treatment of Myasthenia Targets the Physiological Effects and Autoimmune Pathogenesis of the Disease
Anticholinesterases, especially pyridostigmine, provide symptomatic relief, but the treatment is rarely complete and does not alter the basic disease. Immunosuppressive therapies include corticosteroids and azathioprine or related drugs that suppress antibody synthesis.
Plasmapheresis—removing the plasma and the antibodies to the nicotinic ACh receptors or to MuSK—often ameliorates symptoms within days or a few weeks, as does infusion of immunoglobulin.
Although the benefit is transient, it may be sufficient to prepare a patient for thymectomy or to support the patient through more severe episodes. Intravenous administration of immunoglobulins also reduces the titer of antibodies to the nicotinic ACh receptor and to MuSK by mechanisms that are unclear.
There Are Two Distinct Congenital Forms of Myasthenia Gravis
There are two distinct types of myasthenia in which symptoms may be present from birth. In neonatal myasthenic syndrome the mother herself has autoimmune myasthenia that is transmitted passively to the newborn via the immune system. By contrast, in congenital myasthenia the infant has an inherited defect in some component of the neuromuscular junction, rather than an autoimmune disease, and thus does not have serum antibodies to the nicotinic ACh receptor or MuSK.
Congenital myasthenic syndromes fall into three broad groups based on the site of the defect in the neuromuscular synapse: presynaptic, synaptic cleft, and postsynaptic forms. Clinical features common to these disorders include a positive family history, weakness with easy fatigability (present since infancy), drooping of the eyelids (ptosis), a decremental response to repetitive stimulation on electromyography, and negative screening for anti-nicotinic ACh receptor antibodies. A striking feature of many of these diseases is the subnormal development of the skeletal muscles, reflecting the fact that normal function at the neuromuscular synapse is required to maintain normal muscle bulk.
In one presynaptic form of congenital myasthenia the enzyme choline acetyltransferase is absent or reduced in the distal motor terminal. This enzyme is essential for the synthesis of ACh from choline and acetyl coenzyme A (see Chapter 13). In its absence the synthesis of ACh is impaired. The result is weakness that usually begins in infancy or early childhood. In another presynaptic form of congenital myasthenia the number of quanta of ACh released after an action potential is less than normal. The molecular basis for this defect is unknown.
Congenital myasthenia may also result from the absence of acetylcholinesterase in the synaptic cleft. In this circumstance end-plate potentials and miniature end-plate potentials are not small, as in autoimmune myasthenia, but are rather markedly prolonged, which may explain the repetitive response of the evoked muscle potential in those patients. Cytochemical studies indicate that ACh-esterase is absent from the basement membranes. At the same time, nicotinic ACh receptors are preserved.
The physiological consequence of ACh-esterase deficiency is sustained action of ACh on the end-plate and ultimately the development of an end-plate myopathy. This myopathy indicates that skeletal muscle can react adversely to excessive electrical stimulation at the neuromuscular junction. In treating this disorder it is critical to avoid using agents like inhibitors of ACh-esterase that can increase firing in the end-plate and thereby exacerbate the muscle weakness.
Most congenital myasthenia cases are caused by primary mutations in the genes encoding different subunits of the nicotinic ACh receptor. The slow channel syndrome is characterized by prominent limb weakness but little weakness of cranial muscles (the reverse of the pattern usually seen in autoimmune myasthenia, where muscles of the eyes and oropharynx are almost always affected). End-plate currents are slow to decay and channel opening is abnormally long. The mutations probably act both by increasing the affinity of the nicotinic ACh receptor for ACh, thereby prolonging the effects of this transmitter, and by directly slowing the channel closing rate. In some instances quinidine is effective therapy for slow channel syndrome because it blocks the open receptor-channel. As in ACh-esterase mutations, the end-plate degenerates because of excessive postsynaptic stimulation, and thus anticholinesterase medications are potentially dangerous.
In the fast channel syndrome a different set of mutations in one or more subunits of the nicotinic ACh receptor leads to an accelerated rate of channel closing and end-plate current decay. The fast channel syndrome may respond either to acetylcholinesterase inhibitors or to 3,4-diaminopyridine, which increases presynaptic firing and ACh quantal release, probably by blocking a presynaptic K+ conductance.
Lambert-Eaton Syndrome and Botulism Are Two Other Disorders of Neuromuscular Transmission
Some patients with cancer, especially small-cell cancer of the lung, have a syndrome of proximal limb weakness and a neuromuscular disorder with characteristics that are the opposite of those seen in myasthenia gravis. Instead of a decline in synaptic response to repetitive nerve stimulation, the amplitude of the evoked potential increases; that is, neuromuscular transmission is facilitated. The first postsynaptic potential is abnormally small, and subsequent responses increase in amplitude so that the final summated potential is two to four times the amplitude of the first potential.
This disorder, Lambert-Eaton syndrome, is attributed to the action of antibodies against voltage-gated Ca2+ channels in the presynaptic terminals. It is thought that these antibodies react with an antigen in the channels, degrading the channels as the antibody-antigen complex is internalized. Calcium channels similar to those in presynaptic terminals are found in cultured cells from the small-cell carcinoma of the lung; development of antibodies against these antigens in the tumor might be followed by pathogenic action against nerve terminals, another kind of molecular mimicry.
A facilitating neuromuscular block also occurs in human botulism, because the botulinum toxin also impairs release of ACh from nerve terminals. Both botulism and Lambert-Eaton syndrome are ameliorated by administration of calcium gluconate or guanidine, agents that promote the release of ACh. These drugs are less effective than immunosuppressive treatments for long-term control of Lambert-Eaton syndrome, which is chronic. However, botulism is transient, and if the patient is kept alive during the acute phase by treating symptoms, the disorder disappears in weeks as the infection is controlled and botulinum toxin is inactivated.