Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis.

Using pharmacological (Simpson, L.L., 1980, J. Pharmacol. Exp. Ther. 212:16-21) and autoradiographic techniques (Black, J.D., and J.O. Dolly, 1986, J. Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT-induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with 125I-BoNT. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target.

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Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. I. Ultrastructural autoradiographic localization and quantitation of distinct membrane acceptors for types A and B on motor nerves.

The Journal of Cell Biology ◽

10.1083/jcb.103.2.521 ◽

1986 ◽

Vol 103 (2) ◽

pp. 521-534 ◽

Cited By ~ 174

Author(s):

J D Black ◽

J O Dolly

Keyword(s):

Plasma Membrane ◽

Neuromuscular Junction ◽

Nerve Terminal ◽

Motor Nerve ◽

Type A ◽

Nerve Trunk ◽

Metabolic Inhibitors ◽

Motor Nerve Terminal ◽

Electron Microscopic Autoradiography

The labeling patterns produced by radioiodinated botulinum neurotoxin (125I-BoNT) types A and B at the vertebrate neuromuscular junction were investigated using electron microscopic autoradiography. The data obtained allow the following conclusions to be made. 125I-BoNT type A, applied in vivo or in vitro to mouse diaphragm or frog cutaneous pectoris muscle, interacts saturably with the motor nerve terminal only; silver grains occur on the plasma membrane, within the synaptic bouton, and in the axoplasm of the nerve trunk, suggesting internalization and retrograde intra-axonal transport of toxin or fragments thereof. 125I-BoNT type B, applied in vitro to the murine neuromuscular junction, interacts likewise with the motor nerve terminal except that a lower proportion of internalized radioactivity is seen. This result is reconcilable with the similar, but not identical, pharmacological action of these toxin types. The saturability of labeling in each case suggested the involvement of acceptors; on preventing the internalization step with metabolic inhibitors, their precise location became apparent. They were found on all unmyelinated areas of the nerve terminal membrane, including the preterminal axon and the synaptic bouton. Although 125I-BoNT type A interacts specifically with developing terminals of newborn rats, the unmyelinated plasma membrane of the nerve trunk is not labeled, indicating that the acceptors are unique components restricted to the nerve terminal area. BoNT types A and B have distinct acceptors on the terminal membrane. Having optimized the conditions for saturation of these binding sites and calibrated the autoradiographic procedure, we found the densities of the acceptors for types A and B to be approximately 150 and 630/micron 2 of membrane, respectively. It is proposed that these membrane acceptors target BoNT to the nerve terminal and mediate its delivery to an intracellular site, thus contributing to the toxin's selective inhibitory action on neurotransmitter release.

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Steroid anaesthetics: inhibition of depolarization–secretion coupling at the mouse motor nerve terminal

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y80-185 ◽

1980 ◽

Vol 58 (10) ◽

pp. 1221-1228 ◽

Cited By ~ 3

Author(s):

P. Pennefather ◽

E. Puil ◽

D. M. J. Quastel

Keyword(s):

Neuromuscular Junction ◽

Nerve Terminal ◽

Motor Nerve ◽

Motor Nerve Terminal ◽

Nerve Terminals ◽

Potassium Depolarization ◽

End Plate

The coupling between nerve terminal depolarization and quantal secretion of acetylcholine at the mouse neuromuscular junction was estimated by measuring the multiplication of the frequency of miniature end-plate potentials (m.e.p.p.s) produced by increasing the concentration of calcium in the medium from 0.1 to 1.0 mM in the presence of 15 mM potassium. Depolarization–secretion coupling was inhibited by the anaesthetic steroids progesterone, pregnanedione, and alphaxalone. The nonanaesthetic steroid Δ16-alphaxalone also inhibited depolarization–secretion coupling with the same potency as alphaxalone. This result indicates that inhibition of depolarization–secretion coupling in nerve terminals is unlikely to play a major role in the production of anaesthesia.

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Aging increases calcium influx at motor nerve terminal

International Journal of Developmental Neuroscience ◽

10.1016/0736-5748(90)90060-f ◽

1990 ◽

Vol 8 (6) ◽

pp. 655-666 ◽

Cited By ~ 16

Author(s):

Waleed B. Alshuaib ◽

Mohamed A. Fahim

Keyword(s):

Nerve Terminal ◽

Calcium Influx ◽

Motor Nerve ◽

Motor Nerve Terminal

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Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber.

The Journal of Cell Biology ◽

10.1083/jcb.115.3.755 ◽

1991 ◽

Vol 115 (3) ◽

pp. 755-764 ◽

Cited By ~ 37

Author(s):

L Anglister

Keyword(s):

Basal Lamina ◽

Ache Activity ◽

Neuromuscular Junctions ◽

Motor Nerve ◽

Synaptic Cleft ◽

Motor Nerve Terminal ◽

Nerve Terminals ◽

Axon Terminals ◽

Muscle Nerve ◽

Almost All

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.

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