Botulinum toxin is a metabolic product of the Gram-positive, spore forming bacterium Clostridium botulinum; a ubiquitous bacterium, especially present in soil. As a highly e?ective neurotoxin that inhibits signal conduction to the neuromuscular endplate, it is the most potent poison known to man.
The toxin in high doses can cause the disease known as botulism – severe poisoning often acquired by consuming food that has become spoiled and contaminated with botulinum bacteria. The latency period to the onset of symptoms ranges from 4 to 6 hours, but may be up to 14 days in extreme cases.
Symptoms of botulism include:
However, increased understanding of the mechanisms of action of this neurotoxin has led to the therapeutic use of botulinum toxin in modern medicine. Apart from its use in the treatment of various neurological disorders, botulinum toxin has become established as the predominant treatment in aesthetic medicine. It is particularly widely used in the cosmetic reduction of wrinkles, achieved by inducing relaxation of overactive facial muscles.
Structure & Serotype:
Botulinum toxin is a two-chain polypeptide consisting of a light chain (L-chain, approx. 50 kDa) and a heavy chain (H-chain, approx. 100 kDA), which are joined by a disulfide bond. Botulinum toxin can be divided into seven serologically distinct forms, types A to G, with amino acid sequences of the toxins showing a high degree of homology to one another.
The various serotypes differ in their duration of effect and potency, whereby type A has the most potent effect with the longest duration. Thus, type A shows an effect that is about ten times more potent than that of type C, while being as much as 50 times more potent than type B. Type A botulinum toxin is the main serotype in therapeutic use, especially with regard to aesthetic indications. Types B, C and F also play a role in therapeutic applications.
Mechanism of Action:
At the neuromuscular junction, the motor nerve terminal lies in close apposition with the adjacent muscle fiber and induces an excitation-coupling contraction. The motor neuron produces an action potential that travels down the axon to the nerve terminal. Upon the arrival of the action potential, voltage-dependent calcium channels open, causing an influx of calcium ions. This influx results in fusion of the presynaptic vesicles, containing Ach, with the nerve terminal. This fusion is mediated by the SNARE complex (soluble N ethylmaleimide-sensitive factor attachment protein receptor). The SNARE complex is a neural exocytic complex that regulates the membrane docking and fusion of synaptic
vesicles and the release of ACh. The proteins within the SNARE complex include synaptic neuralassociated protein (SNAP-25), syntaxin, and vesicle-associated membrane proteins (VAMP). Botulinum toxin targets these proteins.
When botulinum toxin is administered, the heavy chain (100 kDa) binds selectively to cell membrane receptors on the outer surface of the presynaptic nerve terminal. The entire Botulinum Toxin complex (both light and heavy chains) is then internalized into the nerve terminal via receptor-mediated endocytosis. The vesicles containing the botulinum toxin then fuse with digestive vacuoles that cleave the botulinum toxin molecule into separate light and heavy chains. The light chain (50 kDa) exerts the paralytic effect of botulinum toxin by inactivating the SNARE complex, thereby blocking the release of ACh into the neuromuscular junction. The inhibition of ACh release results in localized muscleweakness (paralysis) that gradually reverses over time.
The mechanism by which botulinum toxininduced muscle weakness is reversed is unknown, but it may involve the intraneural turnover of the affected docking proteins (which are responsible for the release of ACh into the neuromuscular junction), the sprouting of new nerve terminals, or a combination of both of these mechanisms. At 2 months after administration of botulinum toxin, the axon begins to expand, and new nerve terminal sprouts emerge and extend toward the muscle surface. The motor nerve unit is re-established once a new sprout forms a physical synaptic connection with the previous neuromuscular junction.
The new nerve sprouts that do not establish a connection to the motor endplate, however, subsequently regress and are spontaneously eliminated, whereas the parent, or former, nerve terminal is re-established. An understanding of the mechanism of action of botulinum toxin allows one to understand the time required for the onset of paralysis as well as the duration of clinical effect. Botulinum toxin, once injected, takes approximately 3 to 4 days for its effect to become clinically apparent. This corresponds to the amount of time that is required for the botulinum toxin molecule to bind to the motor nerve terminal, undergo internalization via receptor-mediated endocytosis, and block Ach release through inactivation of the SNAP-25 or VAMP SNARE complex proteins.
In contrast, the clinical duration of effect, which is approximately 3 to 4 months in length, corresponds to the time that is required for new unmyelinated nerve sprouts to grow from the nerve root to re-establish the motor endplate, beginning 28 days following injection. The completion of this process occurs approximately 90 days following injection. Therefore, the duration of effect is not dependent on the continued presence of botulinum toxin at the nerve terminal, but rather reflects the length of time that it takes for a particular individual’s nerves to regenerate and develop a functional connection at the myoneural junction.
The heavy chain of the botulinum toxin molecule binds selectively to cell membrane receptors via the heavy chain on the outer surface of the nerve terminal.
The entire Botulinum Toxin complex is then internalized into the motor nerve terminal through receptor-mediated endocytosis. The botulinum toxin type-A is then cleared into separate light and heavy chains. The light chain exerts the paralytic effect by inactivating the SNARE complex proteins, blocking the release of ACh.
The light chain of serotypes A, C, and E exerts its e?ect by cleaving the synaptic neural-associated protein (SNAP-25) that is responsible for fusion of vesicles containing ACh with the nerve terminal cell membrane.
Approximately 2 months after injection, the nerve terminal begins to expand, and new sprouts emerge and extend toward the muscle surface. Additional redundant nerve sprouts are also produced. The motor nerve unit is re-established once a new sprout forms a physical synaptic connection with the previous neuromuscular junction.