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Botulism Paralysed

Botox injection

Botox acts by paralysing small groups of muscles when injected in the face.

By Callista Harper & Frederic A. Meunier

A new class of inhibitors could prevent infection by a neurotoxin classified as a Category A biological weapon.

While the origins of botulism lie in food poisoning caused by contaminated sausages, it is probably best known under its commercial name Botox, which is used in cosmetics to smoothen wrinkles.

However, what is not widely advertised is that botulinum neurotoxin is an incredibly deadly agent capable of killing several million people with just 1 gram. For this reason it is a potential bioterrorist threat, with the USA’s Homeland Security classing it as a Category A biological weapon.

Paradoxically, it is the only Category-A compound that is also approved by the US Food and Drug Administration to treat a wide range of muscular and neurological disorders, and even more for which it does not have official approval.

Although Botox and other brands of botulinum neurotoxins are currently used to treat a wide range of ailments there is still no way of curing cases of abuse or misuse that lead to the disease botulism. This is why it is so invaluable to develop a treatment for this deadly toxin, and why we decided to take a novel approach to this investigation.

Current Treatment Regimes
While on average there is only one case of botulism each year in Australia, compared with approximately 100 cases in the United States, it is still a serious disease with a 5–10% mortality rate. Current treatments are limited and rely on antitoxins and preventative measures such as vaccines.

However, vaccines preclude the use of botulinum toxin as a therapeutic, and antitoxins are only useful if they are administered early while it is still in the bloodstream – before the toxin has had a chance to enter nerve cells. There is also limited availability to the antitoxins, so if a widespread terrorist attack was launched it would be difficult to obtain and administer them to a large infected population in time.

Equine-derived antitoxins are used to treat adults, but due to the increased risk of serum sickness and sensitivity, they are not given to infants. Instead infants are treated with a human-derived antitoxin developed from voluntary donations of serum obtained from vaccinated individuals. However, this is only available from the United States to limited countries and professional personnel (military and scientists). This treatment was successfully used for the first time in Australia in 2010 and, while costing around US$43,500, it is still cheaper than the estimated A$110,000 cost of intensive care required to treat botulism.

Our Results
Botulinum neurotoxins get into the motor nerve cells by hijacking cellular systems that are already in place (see Box 1). Once inside the neurons, botulinum neurotoxins dissociate and cleave key proteins that are necessary for the communication between the neuron and associated muscle.

While most of the current studies are now investigating ways to prevent the toxin from breaking apart and acting inside the nerve cells, we decided to try a novel approach and target the step that botulinum neurotoxin-type A uses to get into cells. To undertake this we inhibited a protein required for endocytosis in neurons in the hope that it would prevent the entry of the toxin. We targeted the protein dynamin, which is necessary for endocytosis and is involved in pinching off the endocytic compartment from the cell membrane.

To achieve this we blocked the function of dynamin with Dyngo-4aTM, a potent inhibitor of dynamin developed recently by Prof Phil Robinson of the Children’s Medical Research Institute and Prof Adam McCluskey of the University of Newcastle. When we tested neurons in culture we found that Dyngo-4a had indeed blocked the uptake of botulinum neurotoxin-type A. We then used different preparations that were more closely related to the natural target of botulinum neurotoxin – the neuromuscular junction – and achieved the same result.

Encouraged by these promising data, we tested the ability of Dyngo-4a to stop the toxin from causing paralysis, and finally used a mouse model to find that Dyngo-4a could actually delay the symptoms of botulism in mice. These results were published in the Journal of Biological Chemistry last year.

Looking Toward the Future
Although these results are only preliminary, we are looking towards more extensive studies to determine the optimal treatment of Dyngo-4a to prevent botulism. The drug is now also undergoing further development to determine its suitability as a therapeutic and to develop more potent compounds.

Our hope is that Dyngo-4a can be used as a prophylactic to prevent the toxin from entering cells. This will allow a larger window of time for treatment with conventional methods such as antitoxins while the toxin is still in the bloodstream.

This approach will be particularly important in the event of a bioterrorist attack, when there is the potential for a large number of casualties and treatments may not be readily available. It is hoped that it could also be used alone or in combination with currently available treatments, which would extend the window of therapeutic effectiveness. Targeting the internalisation step of the toxin should hopefully provide a larger timeframe for treatment, enable treatment of all serotypes and have fewer side-effects compared with the antitoxins.

There is an emerging field that is targeting the internalisation and trafficking pathways hijacked by pathogens in order to prevent their toxic effects. We hope that our study will serve as a stepping-stone for other studies to treat pathogens that also use these dynamin-dependent internalisation pathways to intoxicate our cells.

Box 1: How Botulinum Neurotoxin Acts on Cells
Botulism can result from intestinal, wound or food-borne infections by anaerobic Clostridium bacteria. While these infections are usually due to the toxin entering the body, infants are particularly at risk because the bacteria itself can colonise their intestines and continue to produce the toxin. While incidents have significantly reduced in the past 50 years, several hundred children still die every year from botulism around the world.

Botulinum neurotoxins are highly potent because they can specifically target the motor nerves that control muscle movement. The neurotoxins gain entry into these motor nerve terminals by hijacking existing cellular portals. Many toxins and viruses have developed strategies to “sneak in” via these entry points to access cells and exert their toxic effects, and botulinum is one such toxin that is particularly good at this.

All cells take up signals, fluids, proteins and a variety of other substances from the environment surrounding them through a process called endocytosis. This process allows the cell’s plasma membrane to invaginate and then pinch off to create an internal compartment. One such compartment is a synaptic vesicle that is reformed after it has released a neurotransmitter that enables nerve–muscle communication.

Botulinum neurotoxin binds to glycolipids and proteins located on the membrane of these synaptic vesicles, and the toxin is taken up when the vesicles are endocytosed. A portion of the toxin then drills a hole in the vesicle membrane so that another part can escape and breaks down proteins critically involved in mediating neurotransmitter release. This prevents the communication between the nerve and muscle, and therefore causes a flaccid paralysis that can last up to 4 months. Unless palliative care is sought, this paralysis can rapidly reach the diaphragm and cause death by respiratory failure.

Box 2: The Use of Botox
Botox needs no introduction as a cosmetic, and its use to reduce wrinkles is well-established. It has this effect because it prevents communication between the nerve and muscle, thus relaxing the muscle and reducing the appearance of lines.

Although it does smoothen wrinkles, Botox acts by paralysing small groups of muscles when injected in the face, and therefore reduces facial expression considerably. Thankfully, this effect only last for a few months and facial expression resumes, but so do the wrinkles.

Botox is highly effective because it is so potent and specific in its action. Although it is best-known as a cosmetic, it was first approved for the treatment of blepharospasm (excessive frowning) and strabismus (crossed-eyes) in the 1980s.

Botox is now used for a variety of ailments where the muscles are overactive, such as muscle spasms and cervical dystonia. Botulinum neurotoxin is also used to treat excessive sweating and chronic migraines.

While botulinum neurotoxin is extremely toxic it is a highly effective treatment for a variety of disorders that have few or no therapeutic options.

Box 3: Botulinum Neurotoxin and Bioterrorism
Botulinum neurotoxins have a long history of involvement in bioterrorism. This, combined with its ease of use, has warranted its classification as a Category A biological weapon. The bacteria and its toxins have been developed for both government warfare and bioterrorism for almost 70 years by many countries, including Japan, USA, Germany, the Soviet Union and Iraq. At one stage Iraq had enough toxin to kill the world’s population three times over, some of which was already loaded into Scud warheads.

The Japanese cult Aum Shinrikyo used botulinum in several attacks, and released it into a subway in the 1990s. This group was also responsible for the sarin gas attacks on the Tokyo subway. Thankfully the attacks were unsuccessful, although the reasons for this are unclear.

A/Prof Frederic A. Meunier is a NHMRC Senior Research Fellow and head of the Neuronal Trafficking Laboratory at the Queensland Brain Institute, University of Queensland. Callista Harper is a PhD student in his group.