Botulinum neurotoxins: mechanism of action and therapeutic applications

Botulinum neurotoxins: mechanism of action and therapeut c applications Recent studies have led to the discoveryof the molecular lesions in motor neuronscaused by botulinum neurotox;ns.These neurotoxinsare metalloproteinasesthat enter the cytosol and very specificallycleave protein componentsof the neuroexocytosisapparatus. ConsequenUy, acetylcholine cannot be released and the muscle is paralysed. For this reason, botuHnumneurotoxins are increasingly being used to treat a variety of conditions where a functional paralysis of neuromuscularjunctions is useful as therapy. TOXIGENi(' ~{rai.x {,t Ch~Iridnu~l hottdimtm aad ~rlalcd bacteria prt~lttce pt'olein neuroloxius that cau.~ethe clinical ~yndmme of I~Jttdism. These allaerohic bacteria Call foml sl~res thai are widely distributed in the environment, Seven di!li~rentbotulinum neurotoxins IBoNTs) have so far been ¢l|aracterized (A-G), These nenrolo×ins are active on many ditli~retatypes of vcttebl~ltes~':~uadrileyc~ulpetretmtethe organism 0u~ugh various routes, which determine the three main types of botulism.

Botulism, the disease Foodborne botulism is the most common t'on. in humans, and was first described two centuries ago in Gennany ~'~'following a deadly meal of sausages (the name botulism derives from the latin word i~,' sausage, botulus). (?loslridimn botulbmm, the neumtoxin-producing bacterium, was identified a century later, in Belgium, |ollowing another episode of botulism caused by the ingestion of contaminated meat. More than 12000 cases of foodbome botulism have been recorded since 1951 worldwide, and they are associated with three serotypes (34% to type A, 52% to type B and 12% to type E: only two cases were associated with type F)''. The disease results from the ingestion of Sl~re.contaminated toods that have been maintained in anaerobic conditions, allowing germination, proliferation and neurotoxin production, BoNT survives passage through the acid and proteolytic environment of the stomach, protected by accessory proteins (Fig. I). In the intestine, it dissociates from these proteins, crosses the intestinal epithelial layer and spreads throughout the o~anism. Foodbome botulism is therelbre an 'intoxication" rather than an infection, even though sometimes its epidemiology resembles that of /

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an hffecfious disease ~:. The incidence of foodbomc

botulism is dcctinmg as a coaseqae~cc of mq~mvcd food preservation procedmc~ A second lbnn of the disease, inihn~ bo~tdism, ~as recognized as a distinct clinical entity only 20 yea~ ago~; 1134 cases have been recordeds worldwide up to 1994. and in the USA i~ is currently the most frequem feral of bolulism. In lhe neonate's imesfine, neumloxigenic Clostfidia can gemainale from spores and proliferate in the absence of a competing bacterial flora, which would otherwise prevent theh growth, Thus, infant botulism is a toxic infection ~. A third and much ~ess frequen~ form is wound botulism, u disease flmk like tetanus, resuhs fl'om the contamination of wounds with bacgerial spores m~'. Massive fatal outbreaks of botulism are not uncommon in animals, particularly among birds and fishes, both in tile wild and on farmsL-"L Tile signs and symptoms of botulism are essemiaHy the same in ali fom~s of die disease, and are nol as striking as those of tetanus, a neuroparalytic disease caused by a neurotoxin that is structurally very similar to BoNT. This could account for the more recent recognition of botulism as a disease"S compared with tetanus, particularly lee infant botulism ~. Symptoms usually develop 12-36 h after food ingestion and are all related to a sustained blockade of acetylcholine release al somatic and autonomic nerve terminals ~~. Muscles controlling vision, swallowing, brealhing, neck movements and posture are affected. Dryness of the rnoulh and pharynx is also found, and some paticuls report other autonomic effects such as nausea, vomiting and abdominal pain. hflhnt botulism is characterized by lethargy, listlessness, feeble cq¢, pOOl Iccdhig, weakness, hypatonia, plosis and apnoea. Mosl palients stirrive, allhough recovery is OIlel| Slow {recovery time i.,, longer after BoNT/A than after BoNT/B intoxication); however, some botulism patients die, mainly fi'onr res.pimtory or cardiac arrest. The Cald'nmiu Deparlmem of Health Services at Berkeley has developed a hum:u)derived anti-h)tulinum neurotoxin antiserum; a clinical trial of its use Ibr infimt botulism is currently under way and is expected to be completed within a few years (contact Dr. S.S. Amen, infant Botulism Prevention Program, California Dept of Health Services, Berkeley, CA, USA). The structure of botuLinum neurotoxins All the clinical symptoms of botulism are caused by BeNT itself, as they can be induced by injection of the purified toxin. Moreover, animals can be protected from botulism by immunization with chemically detoxified botulinum neurotoxins. A pentavalent botulinum toxoid vaccine has been developed to protect humans against BoNT/A. /B, /C, /D and /E. This experimental vaccine has a moderate efficacy and is available through the Centers for Disease Control and Prevention (Atlanta, GA, USA; reviewed in Ref. 6). BeNT is active on all vertebrates, although the BeNT

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Rgure1. Structureand mechanismof activationof botulinemneuroloxins(BoNTs).(a) The toxins are releasedby bacteriaas progenitortoxins(extra large,LL), composedof the toxin polypeptide chain plus one or more copies of accessory proteins: nontoxic non-haemagglutinin protein (NTNA, purple) and haemaggMinin protein (HA, brown), which increasethe resistanceof the toxin to acids and proteinases. BoNT/A used in human therapy is in the form of progenitor toxins, because it does not lose activity upon lyophilization.(b) In mildly alkaline media, such as extracellularfluids or the sterilephysiologicalsolutionsused to dissolvethe tyophilizedtoxin, progenitortoxins(large,L) dissociateand (©)releasethe inactivetoxin(medium,Mr. (dr This 150 kDa toxin is activatedby selective proteolysis,which may take place before dissociationof the progenitortoxin,to term (e) a di-chaintoxincomposedof a heavychain (H, 100 kDa) and a lightcha=n (L, 50 kDa) linked by a single disulphidebond. Botulinum neurotoxinsconsist of three 50 kDa domains, which play differentfunctional roles in cell penetration:Hc (lightblue), the C.terminal half of the H chain, is involvedin neurospecificbinding, whereasH. (green),!he N-terminalhall of the H chain, is involved in cell penetration, (f) Reduction takes place inside the nerve cell and liberatesthe 50 kDa L domain (red) in the ¢ytosol. L is a Zn~'-dependentmetalloproteinase specificfor three protein components of the neuroexocytosisapparatus,The structure of progenitor toxins is based on Ref, 38 with the kind permissionof Prof. K. Oguma. I

4]9

types have varying potencies in different animal speciest:'~. They are the most poisonous substances known for mammals, and their toxicity depends on the route of administration: injected BeNT is more toxic than orally administered BENT. The dose that kills 50% of mice (MLD~) is 0.1-1 ng kg- ~for all serotypes; the lethal dose for humans is estimated to be in the same range. As depicted in Fig, 1, BoNTs are produced as inactive, single polypeptidechainsof 150 kDa. They are releasedby bacterial autolysis as complexes with other nontoxic proteins, and these complexes are termed progenitor toxinsL~'7'~.Three forms of progenitor toxins have been identified~ far:e~ttra-largesize (LL, sedimentsat 19S, +900 kDa); large size (L, sediments at 16S, 500 kDa) and medium size (M. sediments at 12S, 300 kDa), BeNT dissociates from its accessory proteins at the moderately alkaline pH values of the intestine. BoNTs complexed as progenitor toxins are more stable than isolated BoNTs to proteolysis and denaturation induced by temperature, solvent removal or low pH (Ref. 4). Several clostddial and tissue proteinases can cleave single-chain BoNTs at an exposedloop and generatean activedi-chaintoxin (Fig. 1).

Glossary ApnoR - The cessation or suspension of breathing. Oiplopla - A sight disorder in which one object is perceived as

two, I,e, douhla vision, Dyeefthde - Impairment of articulation caused by any disorder or lesion affecting the tongue or speech muscles, Oyt~noes ~ Difficultor laboured breathing. Oyltonla - Any disorder of muscular tonleily, N e u r o e K o ~ i t i = The cellular proco~ by whtch neurotransmltter~ or neuropeptides, contained inside syn~,ptlc veslcle~ ~t nerve tar. mlnals, are released in the intersynapttc space following Ca -~+. Irlduced vesicle fusion with the plasma membrane, I~NIS = Falling down of an organ or pert, e,g, drooping of the eyelid, Polyekdoglutgliolddes - Uplda composed of two hydrocarbon chains linked to a saccharide, highly abundant at nerve terminals. Vetlcle-eseocllaed membrane protein (VAMP) - Also termed syneptobrevln, a 12 kDa protein anchored to the membrane of cell vesicles; it is abundant on the neurotransmitter-containlng small synoptic vesicles and Is essential for neurotransmtttorrelease, Synapto~mHuw~'.lated membrane protein of :~S kDe (SHAPe§) - A protein bound to the cytosolic face of the nerve plasma membrane via palmtteylation of cysteine residues; it is involved in neureexoeytesis as well as in axonal growth, - A state of Increased muscular tenus tending to involve the flexors of the arms and extensors of the legs, associated with exeg~mted tendon reticle and a speclltc two-phased pattern of m~ular response to stretch, Synte~dn - A 30 kOa protein located on the ¢ytoselic face of cell membranes, essential for neuroexocytosis as well as for other events of vesicle fusion with a target membrane.

m

429

The heavy chain (H, 100 kDa) and the light chain (L, 50 kDa) remain disulphide linked. Availableevidence indicates that BENT: :onsist of three 50 kDa domains (see Refs 9, 10 for detailed discussions and references) (Fig. 1). This ~tmcture--functionorgan;zation is related to four distinct steps of the ceil-intoxicationmechanism,depicted in Fig. 2. They are: (1) binding, (2) internalization, (3) membrane translocation with reduction of the interchain disulphide bond and (4) intracellular catalytic activity, which is displayed by the L chaint° (Fig. 2). The C-terminal half of the H chain (Hc) is mainly responsible for the binding to neuronal cells, whereas the N-terminal half of the H chain (HN)is implicated in the membrane lranslocation of tim L chain. Toxin binding and penetration into motor neurons After diffusion in the body fluids, BoNTs bind selectively to an as yet unidentified presynaptic component(s) of the neuromusctdar junction (NMJ) (reviewed in Ref. it)). Recently, BoNT/B was shown to bind to the N-terminus of synaptotagmin II complexed with gangliosides~t but the different BoNTs appear to bind to different nerveterminal receptorsm3. These receptors must bind the BoNTs with very high affinity since MLD.~o values correspond to very low molar concentrations. Bindingto isolatedNMJs is rapid, and can occur at low temperatures and in the absence of nerve stimulationE~.it is possible that part of the variability in the response to BoNT/A injection among patients can be attributed to subtle differencies in the number and/or exposure of neurotoxin receptors. As long as the toxhi ;~; bound :o the plasma membrane and exposed to exti'acellularfluids, it can be neutralizedby antibodies against BENT, but after internalization into neurons, the toxin is no longer accessible to antibodies. This limits the time window of efficacy of immunotherapy in the treatment of botulism to a few hours after BeNT has entered the organism, except tbr infant botulism where there is continuous toxin p, oduction in the infantine. To reach their site of action in the cytosol, BoNTs must cross tlae hydrophohicbarrier of die vesicle nlembrane (Fig, 2c). This membrane translocalion step is common to all bac|erial toxins wilh intracelhdar targets, and is the lenst uuderstood step of the entire cell,intoxication process, Pharmacological evidence indicates that acidification of the lumen of the BeNT-containing intraceilular vesicles is required for entry of the L chain to the cytosol'a (see Fig. 2), How bot~linum neurotoxins block Ihe release of acetylcholine BoNTs are zinc-~.oatainingproteins that bind one Zn:' ion to the His-Glu-x-~-His zinc-binding motif, which is characteristic of all metaUoproteinases|s. BoNTs are highly specific, they only recognize and cleave three protein components of the apparatus that mediates neurotransmitter release at synoptic terminals (references in ReE 10: Fig, 2), The core of this apparatus is composed of vesicle-associated membrane protein (VAMP, also known as synaptobrevin;a plotein of the nenmtransmitter-containingsmall synopticvesicles),and two proteins of the cytosolic surface of the presynaptic membrane: synaptosomeassociated membrane protein of 25 kDa (SNAP-25) and syntaxin~''7. These three proteins form a heterotrimeric complex|~ that serves as a scaffold for the assembly of the apparatus~9, Ca:' entry at the nerve terminal triggers membrane fusion, and neurotra,'~smitteris released within a few hundred microseconds. In the cytosol, the L chains of BoNT/A and/E hydrolyse SNAP-25, whereas BoNT/td, /D, /F and /(3 cleave VAMP, and BoNT/C cleaves both SNAP-25 and syntaxin, causing death of neurons in culture't-'-~. Each one of the different

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Figure 2. Current view el botulinum neurotoxin(BENT)entry at nerve tem/inals. This ligure depicts a ~cenario tot BeNT entry into neuronsIhat is m agreement with all availableexperimenlaldata. (a) BeNTbinds to the presynapticmembraneat as yet unidentified receptors(purple)in the neuromusculariunction (NM,I) (b) Binding i~iIollowed by in|emalizalioa lnsidg an endocyticvesicle, which is ({:) acidilied by a vacuolarATPa~oproton pump (H') (d) At low OH, BeNTcl~angos~;on, formation and can new inserl into the lipid bilayer el the vesicle and tran,~localetire 50 kOa L cllain into the cytosol, (e) In.sidelhe cyto~.ol,the L chain (red) catalyses Ihe Zn~'-depondentproleolysis el one oi the protein componentsof the nouroexocytosisapparatus BoNI/B,/O, ,'F and/G attack VAMP/synaptobrevin,a protein of synapiic vesicles, whereasBoNT/Aand/E cleave SNAP-25and BoNT!Ccleaves both SNAP.25and synlaxin, two proteins of tl~ecylosolic !ace of the preeynapticmembrane.TI'e result of the cytosoltccatalytic aclivtty el the L chain is a persistentblockadeo| acetylcholine(ACh) release~=~dhencean impairment of muscularcontractionbecauseIhe muscleacetylcholtnereceptors(AChR)are not activatedby ACh,

BoNTs catalyses the hydrolysis of different peptide bonds (see Ref. 10 for details and references). As a result, the cleaved proteins cannot perform their functions and neuroexocytosis is prevented. There are three isoforms of VAMP (VAMP-I, VAMP-2 and cellubr~.vin), which ~ne present in all cell ~ype:,tested ''n. BoNTs have no effect on non-neuronal cells owing to the absence of surface receptors that mediate toxin entry. However, other factors might also be involved in resistance to clostridial neurotoxins. It is known that chickens and rats are not sensitive to either BoNT/B or to tetanus neurotoxin, and it has been suggesterl that the single amino acid substitution at the site of cleavage of VAMP-I by these two toxins (a reline replaces the glutamine residue present in mice and humans, both BeNT/B-sensitive species) is responsib!e for ::uch speciesspecific resistance 2~. SNAP-25 is a 206-residue protein required for axonal growth during development and possibly also nerve-terminal plasticity in the

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mature nervous system21'z4:s. Two human SNAPo25 isoforms are known, which are equally sensitive to BoNT/A and/E, and are differ~ entially expressed dunng development. Several isoforms of syntaxin have also been iden|ified r~ and all its predominant neuronal isofom~s are cleaved by BoN'F/C ~'r;. Sy~llaxin is present at plesy~mptic at,ire :. n:.;, where neurotransmitter release takes place, in association with the C:P channels that mediate the entry of Ca :+ upon membrane depolarization ~7. The membrane topology of syntaxin is similar to that of VAMP: most of it is expo.~ed Io the cytosol and tethered by a C-terminal membrane anchor. The L chains of the seven BeNT types are very similar and have e,,olved from a common ancestor~'t", but the differem types recognize and cleave three different targets (Fig. 2). The specific recognition of VAMP, SNAP-25 and syntaxin is largely determined by BeNT binding to a nine-residue segment present only in these three proteins ~°.zS. 421

Therapeutic u~s of botulinum neurotoxins The demonstration by Burgen and colleagues '~"that BoNTs block acetylcholine release at the NMJ, and subsequent studies on the histological effects of BoNT in animal muscles (reviewed in Ref. 30)

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provided the basis for the use of these toxins as therapeutic agents ~t. However, a quarter of a century elapsed before Alan Scott, in search of an agent able to elicit a functional paralysis of ocular muscles. began to study BoNT/A~'. Following the publication of his first study on strabismus ('crossed eyes') in 1980 (Ref. 33), papers reporting the clinical use of BoNTs largely outnumber those dedicated to botulism or to the cellular biochemistry of BoNT. Nowadays, BoNTs are used in the therapeutic management of several focal or segmental dystonias, of strabismus, and also in any situation where a reversible depression of a particular neuromuscular function is desired (see Table 1). Injection of BoNT into a mammalian striated muscle causes a variety of histological changes (reviewed in Ref, 301. A first sign is the accumulation of synoptic vesicles on the cytosolic face of the plasma membrane "~'~".This is the immediate consequence of the pro° teolytic cleavages discussed above: ~,esicles callao! discharge theh neurotransmitter content and hence cluster on the plasma membrane. Contrary to what happens in denervation obtained by other means, such as nerve ligation, anatomical contacts between the nerve and muscle are maintained and there is no apparent loss of motor axons. The motor end plate enlarges and, under the effect of growth factors released by muscles, sprouts devdop from the end plate itself', from tile ten!final part of the axon and from the nodes of Ranviea. and grow into tile muscle, The number of motor end plates on a single muscle fibre also increases. Axon collaterals develop and lead to an increase in the number of fibres innervated by a single motor axon. Moreover, it is possible to identify some muscle fibres that are innervated by more than one motor axon. The alterations seen in BoNTtreated muscles parallel those documented in olher forms of denervalton, Muscle fibres u||dergo a progressive atrt~phy with reduction of their mean diameter, which begins in the first two weeks after BoNT injection and pt~gresses for ~ 6 week:;. Acetylcholinesterase and ucetylcho!ine receptors spread from the NMJ over the muscle plasma ntelnbrane, Following axon~d spn~nting anti eeoformalion of Innctional netve.qnuscle junctk~ns, the muscle eve,~tually regains its normal size aitd both acetylch|}lh|esterase and acetylcholine iccep|~rs l~ct~ncen~ tr~!te nt !he NMJ alt,le. The muscle atrophy induced by BoNT in ani~ real models and in humans is therelbre largely reversible, even alter repeated BoNT injections '~',The number of morphofunctioual studies devoted to the effects of denervation with BoNT is, however, limited and has mainly been obtained from animal models; few studies have analysed human specimens (references in Pet'. 30). Tlese e~perimenlal studies and the analysis of the follow-up of the therapeutic use of BoNT indicate that the functional recovery of neuvnmuscular activity after BoNT injection depends on the type of muscle injected, the dose of BoNT and the BoNT serotype used. The therapeutic protocols currently available were developed largely on the basis of clinical practice and vary considerably between centres. One aspect of such an experience should be emphasized: the extensive use of this very toxic substance in different centres is accompanied by a r~'markable and common record of sat'ety~. Experience has accumulated primarily for BoNT/A and little has been reported on the other types of BoNT. Cie~u'iy,BoNT as a therapeutic agent is still in its infancy ~uld its lull therapeutic putential remains to be determined (see Box I), Howevei. tilere are some developments that can be anticipated.

Standardi:ation of clinical protocolsfor the therapeut&use of BoNTs The amount of toxin to be used currently varie:; depending on the size of the muscle to be treated and on each patient. The dose to be m

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o Standardization of clinical protocols and toxin preparations o Deliveryof smaller toxin doses very close to the neummusc~ar junction o Testing o~ toxin types olher th:m BeNT/A on tt~e~r own or as cocktails of t,a'oor more toxins ® Developroent of route potent toxins wish prolonged action ~d lower imrounvgenicity Extension of clinical use of tmtulinum neumtoxins go other pathological conditinns in oalmpaedics, playsiotherapy, plastic and cmmetie surety

used should be estimated for each patient by can3,ing out a preliminary test in the extensor digitoris brevis muscle of a toe ~a. The nun> bet, volume and sites of injection that provide the optiroum resuits for each clinica~ application also need to be agreed.

Fm'ther det,elopmems of BuNT The toxin currently used is ,'t complex of goNqTA ~ifi~ its accessory proteins (Fig. I). This complex is more stable than the pure toxin and better survives lyophilization, ltowever, a large variation in potency among differen! batches is found, and most of tile protein present comprises BoNT/A accessory proteins. The presence of an inactive, but immt.nogenic, toxoid form of BuNT should also be collsidefed. The major drawback of repeated trcatments with BuNT/A, pmaiculady of latge muscles, is the fonnatkro of anlibodies to the toxin complex, which renders a patient unresponsive go further BoNT/A treatment, Tl~erefore, |oxoid l'omls of BuNT have to be eliminated. It is uko passible thai accessory proteins are immtmo=adjuvaut with respect to tile Iol|nulion of atnlibndies against BoN'E lit the pats| two years, we trove Iiealed many w)[unteers aim pulients with prep;u'tritons o1' BoN'IYA, tB,/C and/F that at~e free of accessory proteins and have not yet recorded any negative effect, or de|ecled immunnglobulins against BuNT (R, Eleopra el a/., unpublished). Anolher potential improvement regards the L chain of BUNT; this is tile metalloproteinase that cle:wes SNAP-25, VAMP or syntaxht. The re-establishment of a functional synapse can be assumed to Iollow the inactivation of the L chain in the neuronal cytosol and the reformation of physiological levels of these proteins as a synaptic pool. Modification of the L chain, by either genetic engineering or chemical manipulations to prolong its action within the ceil, should extend the duration of tile benefical effects of therapy. A contplementary approach is that of interfering with the processes of sprouting and reestablishment of a functional N MJ.

"~-~ ~:' ~" ~ @ ~ :

ncumflizc the ~oxia cat~ iotas. (>,her BoNi ~ype~ are ~o'a ava',[ b!t for cfinicai use to circumven~ the probIem of circu~afi~g arttibe o~r own experiences, indicate that the original symptoms reappear faster alter treatmertt with BoNT¢5 or BuNT/B, compared with BuNT/A: tt~is is in agreement with the faster recovery time from BoNT/B-botnlism. This resuh can be attributed to differenl causes, such as the L chains of" BoNT/B or/F having a shorter ac6ve life within the neuronal cytosok or a faster resynthesis of VAMP with respect go SNAP-25. A resent study with cortical neurons in culture -'t suggests ~hat this could be related to the essential rote of SNAP-25 in axonal growth, a process unaffected by the BoNT/B and/F-sensitive VAMP. The fan that BoNT/C cleaves two proteins of lhe nem'oexocytic machinery"" ,e suggests that this toxin might cause a longer-lasting paralysis than BoNT/A. BoNqTC was recently tested ol~ the human extensor digitoris brevis muscle of file toe and was found to be veu efficacions, despite the fact that BoN%C is usually associated with botulism in animals other than hronans'-~. The paralysis induced by BoNT/C [ast~ us ior~g as that caused by BoN'ITA {R. Eleopra et al., unpunished), and BoNT/C would seem to be a valid and promising alternative to BuNT/A, worthy of future examination. Our prdiroinary experience with patients affected by blepharospasro and heroifacial spasm ful b supporl this propos,'d (R. Eleopra eta/., unpublished)

Ofker clinical uses There are a variety of conditions tha| could benefit fl'on| treatment with BoNT. Apart from aesthetic uses. such as the treatment of facial wrinkles ~'' or the con'eclion of assymmelry arising f,'oro unik~teral rousde paralysis (Fig. 3), BuNT appears go be usefl:' to prevenl ilk' undesmtble effects of hypercontracfion h| many rousc[es, h~jection of BoNT/A in the |nolo-anal ronscie to prevent inrmafion eft anal

Other toxins and toxin combinations So far, BoNT/A has been used most olien lor therapeutic purposes (Ref. 32 and references cited therein}. In a few cases, BoNT/A treatment is not effective on the first attempt. The reason(s) for these failures have not been investigated, but pre-existing antibodies consequent to immunization during a subclinical episode of botulism might contribute to this unresponsiveness. Alter repeated injections of the high doses necessary to treat large muscles, particularly when the toxin is injected in anatomical areas rich in lymph nodes, antibodies that

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Figure 3. Use of botulinum neurotoxin for an aesthetic application. This patient had surgery for the removal of a parotid gland tumour, which caused a unilateral facial paralysis and a profound compression of the lower portion of the ~ace. (a) After surgery to remove the tumour, I~er facial posture was asymmetrical. Botulinum neurotoxin type A [oculinum (Allerghan), 20 IU] was injected into three muscles - risorius, buccinator and depressor labi inferior (or nearby muscles) - et three-month intervals. (b) The photograph was taken 22 months alter the first treatment with BoNT/A

i 423

M()I,I]CLJI_.AR M I ~ D I C I N I ~ ' | ' ( ) D A Y , OCT()I~IEI,~ 1(4o0

fissures~ is an example of this, but its potential in the prevention or treatment of other pathological conditions related to cholinergic functions has yet to be explored (Box 1). This potential extension of the clinkal use of BuNT appeals both to clinicians who are beccm ing involve ~ in the deue!opment of this novel therapeutic tool, and to the pham~aceutical industry: four companies have already developed BuNT/A,/13 and/F for the treatment of dystonias and strabismus.

13 Dolly, J.O., Black, J., Williams. R.S. and MeUing, J. (1984) Acceplors for botnlinum nearotoxin reside on motor nerve terminals and mediate its internalixarian, Nature 307. 457--460 ~', ~;i~pson, L.L., Coffield, J.A. and Bakry, N. (1994) |nhibition of vacuolar adenosine triphosphatase antagonizes the effects of ¢lostridial neurotoxins but not phospbolipase A~ neurotoxins, J. Pharmacol. Eap. Thee. 269. 256--262 15 Barrett, A.J., ed. (1995) Proteolytic Enzymes: Aspartic and Merallopeptidases (Methods in Enzymology Vol. 248), Academic Press 16 Rothman. J.E. (1994) Mechanisms of intracellular protein transport, Nature 372, 55-63 17 Sudhof. T.(' (tOt)~) The ffnaptic v~icle cycle: a cascade of protein-protein interactions, Nature 375,645-653 ® b it possible tour~ the heavy chain of ¢ ~ ~xim 18 Hayashi, 1'. et al (1994) Synoptic vesicle membrane fllsion complex: action of to deliver blolog~als, such as ~nzTmes or antibodies, to nerve closIridlal neurotoxins on a.~emb|y. EMBO J, 13.5f)51-506| ~lls? 19 Pellegfini, L.L. et aL (1995) Clustridtal nema)toxlns con|promise the stability of q~ How do b~tuliuum neurotoxins bind and enter peripheral a low energy SNARE complex mediating NSF activation of synaptic vesicle motor n~umm? fusion, EMBOJ. 14, 4705-4713 • How can the toxin be delivered to specific netm~mt~u~u" 20 Foran. P. et aL (1996) Botulinum nenrotoxin C[ cleaves both ffntaxio and junctions and how can its entry into the motor neuron cytosol SNAP2$ in intact and permeabilized chromaffin cells: correlation with its bo increased? blockade of catecbolamine release, Biochemistry 35. 2630-2636 Whydo some peopLe not repond to treatment whh d~ 21 Osen.Sand. A. e: aL (1996) Common and distinct fusion.proteins in axonal neurotoxin? growth and transmitter release. J, Camp. Neural 367, 222-234 • How do botu!iaumneutmaxins in bondismc ~ the m ~ 22 WilliamsonoL.C. et al. (1096)Closlridial neurotoxlns and substrata proteolysis In intact neurons: botulinum neurotoxin C acts on SNAP.2$, J Biol. Chem 271, 7694.-7699 23 Patamello, T, et al, (1993) Neutotransmission and secretion, N~tnre 364, 581~582 Aeknuwl~geme, t~, W~ apologize to those colleagues who~ papers could not be 24 Oyler. G.A. et al. (1989) The identification of a novel synaptosomal.associoted cited o~i~g to a restriction on space. Work of C,M, is partially supported hy Telethon. protein, SNAP.25, differently expressed by neanmal subpoimlafions, J. Celt Italia gram 763, Biol, 109, 3039-3052 .)S O~l~-Sand. 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