Scientists solve mystery of how Botox attacks nerves

Every year, millions of people try to look younger by taking injections of Botox, a prescription drug that gets rid of facial wrinkles by temporarily paralyzing muscles in the forehead. Although best known as a cosmetic procedure, Botox injections also have been approved by the Food and Drug Administration (FDA) to treat uncontrolled blinking (blepharospasm), lazy eye (strabismus), involuntary muscle contractions in the neck (cervical dystonia) and acute underarm sweating (severe primary axillary hyperhidrosis).

Botox users might be surprised to learn that they're actually receiving minute injections of a bacterial neurotoxin called botulinum, one of the most poisonous substances known. Exposure to large amounts of botulinum bacteria can cause a paralytic, sometimes-fatal disease called botulism. Last month, several Floridians were hospitalized with botulism after receiving injections of an anti-wrinkle treatment that authorities suspect was a cheap, non-FDA-approved imitation of Botox.

The botulinum toxin works by invading nerve cells, where it releases an enzyme that prevents muscle contraction. In recent years, scientists have determined that the enzyme binds to specific sites on proteins called SNAREs, which form a complex in the synapse between nerve and muscle cells. Without SNAREs, nerves cannot release the chemical signals that tell muscle cells to contract, and paralysis results.

"The botulinum enzyme selectively attacks one of the SNARE proteins and cuts it into two pieces," said Axel T. Brunger, a Howard Hughes Medical Institute investigator who holds professorships in three Stanford departments—molecular and cellular physiology, neurology and neurological sciences, and the Stanford Synchrotron Radiation Laboratory (SSRL). "That's sufficient to disrupt its function. But the means by which the enzyme identifies and cleaves its target SNARE has been a subject of much speculation."

Now, Brunger and graduate student Mark A. Breidenbach have solved part of the puzzle. Their results, published in the Dec. 16, 2004, issue of the journal Nature, could help researchers develop alternative treatments for botulism and perhaps find new medical applications for Botox and other neurotoxins.

Clever machinery

There are seven forms of botulinum neurotoxin produced by seven different strains of the Clostridium botulinum bacterium, explained Brunger. "The seven botulinum neurotoxins cut SNARE proteins at different sites along the surface," he said. "Why that is, we really don't know exactly."

For the Nature study, the researchers focused on one of the seven forms, botulinum serotype A, which is the active ingredient in Botox. Breidenbach, lead author of the study, spent months in Brunger's lab trying to crystallize a SNARE/botulinum A complex for laboratory analysis. Unfortunately, the botulinum A samples usually ended up slicing the SNARE target in two, rendering it useless.

"The trick that Mark found was to introduce two specific mutations in the botulinum enzyme that would inhibit its function, but not to the degree that it would affect its structure," Brunger said. "These two mutations prevented it from cutting, so we could observe how it interacted with an intact SNARE."

The SNARE/botulinum A crystals were then taken to SSRL and the Lawrence Berkeley National Laboratory, where their structures were determined using a technique called X-ray crystallography. The results, published in Nature, reveal a complicated, three-dimensional maze of twisted proteins that look like gift-wrapping ribbons gone awry.

"What we've shown is that part of the targeted SNARE protein literally wraps itself around the botulinum A enzyme, using a large portion of the enzyme's surface for specific interactions," Brunger noted. "That's the novel finding in our study."

It turned out that the SNARE protein was actually bound to more than two dozen sites on the enzyme. "Such an extensive interface between a neurotoxin and its target is unheard of," Brunger said. "What botulinum A has accomplished with this large interaction area is a high degree of specificity with just a single unit. Often in biology such specificity is accomplished by having large complexes of auxiliary proteins working together, but these bacteria use a very simple mechanism—in this case, a single protein. It's an extremely clever machinery."

New applications

Brunger hopes to determine the structures of other botulinum enzymes, along with a closely related neurotoxin that causes tetanus, another serious muscular disorder that affects hundreds of thousands of people worldwide every year.

"Perhaps one could develop drugs that would treat botulism and tetanus by competing with specific binding sites on the surface of the neurotoxin," he said. "The idea is that you could inject people with a compound that would have an immediate effect."

Further research also could open the door to novel medical applications, Brunger added. For example, recent experiments have shown that Botox may be useful for treating ringing in the ears (tinnitus), urinary incontinence and excess scarring that occurs when a wound heals.

"This whole field is very young and evolving, and the picture we have so far is incomplete," he concluded.

The study was supported in part by the U.S. Department of Energy and the National Institutes of Health.

Contact:

Mark Shwartz, News Service: (650) 723-9296,
mshwartz@stanford.edu

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