Botulinum neurotoxin type A: actions beyond SNAP-25?

Botulinum neurotoxin type A (BoNT/A), the most potent toxin known in nature which causes botulism, is a commonly used therapeutic protein. It prevents synaptic vesicle neuroexocytosis by proteolytic cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25). It is widely believed that BoNT/A therapeutic or toxic actions are exclusively mediated by SNAP-25 cleavage. On the other hand, in vitro and in vivo findings suggest that several BoNT/A actions related to neuroexocytosis, cell cycle and apoptosis, neuritogenesis and gene expression are not necessarily mediated by this widely accepted mechanism of action. In present review we summarize the literature evidence which point to the existence of unknown BoNT/A molecular target(s) and modulation of unknown signaling pathways. The effects of BoNT/A apparently independent of SNAP-25 occur at similar doses/concentrations known to induce SNAP-25 cleavage and prevention of neurotransmitter release. Accordingly, these effects might be pharmacologically significant. Potentially the most interesting are observations of antimitotic and antitumor activity of BoNT/A. However, the exact mechanisms require further studies

Botulinum toxin type A in motor nervous system: unexplained observations and new challenges

In the motor system, botulinum toxin type A (BoNT/A) actions were classically attributed to its well-known peripheral anticholinergic actions in neuromuscular junctions. However, the enzymatic activity of BoNT/A, assessed by the detection of cleaved synaptosomal-associated protein 25 (SNAP-25), was recently detected in motor and sensory regions of the brainstem and spinal cord after toxin peripheral injection in rodents. In sensory regions, the function of BoNT/A activity is associated with its antinociceptive effects, while in motor regions we only know that BoNT/A activity is present. Is it possible that BoNT/A presence in central motor nuclei is without any function? In this brief review, we analyze this question. Limited data available in the literature warrant further investigations of BoNT/A actions in motor nervous system

Effects of botulinum toxin type A facial injection on monoamines and their metabolites in sensory, limbic and motor brain regions in rats

Despite its toxicity, botulinum neurotoxin type A (BTX-A) is a valuable therapeutic agent for several motor, autonomic and pain disorders. Numerous studies have described its peripheral as well as central effects. Using reversed-phase High Performance Liquid Chromatography with Electrochemical Detection (HPLC-ED) and gradient elution, we quantified the concentrations of dopamine (DA), noradrenaline (NA), serotonin (5-HT) and their metabolites in 10 brain regions, ipsilateral and contralateral from the site of unilateral BTX-A administration (5 U/kg) into the rat whisker pad. In regions associated with nociception and pain processing we also examined possible BTX-A effects in combination with formalin-induced inflammatory orofacial pain. The dominant BTX-A effects on the monoamines and their metabolites were insignificant. The only significant increase caused by BTX-A alone was that of NA in striatum and serotonin in hypothalamus. While antinociceptive effects of BTX-A are most probably not related to central monoamine concentrations, the localized increased NA and 5-HT concentrations might play a role in reported BTX-A efficacy for the treatment of depression

Central Action of Peripherally Applied Botulinum Toxin Type A on Pain and Dural Protein Extravasation in Rat Model of Trigeminal Neuropathy

BACKGROUND: Infraorbital nerve constriction (IoNC) is an experimental model of trigeminal neuropathy. We investigated if IoNC is accompanied by dural extravasation and if botulinum toxin type A (BoNT/A) can reduce pain and dural extravasation in this model. ----- METHODOLOGY/PRINCIPAL FINDINGS: Rats which developed mechanical allodynia 14 days after the IoNC were injected with BoNT/A (3.5 U/kg) into vibrissal pad. Allodynia was tested by von Frey filaments and dural extravasation was measured as colorimetric absorbance of Evans blue - plasma protein complexes. Presence of dural extravasation was also examined in orofacial formalin-induced pain. Unilateral IoNC, as well as formalin injection, produced bilateral dural extravasation. Single unilateral BoNT/A injection bilaterally reduced IoNC induced dural extravasation, as well as allodynia (lasting more than 2 weeks). Similarly, BoNT/A reduced formalin-induced pain and dural extravasation. Effects of BoNT/A on pain and dural extravasation in IoNC model were dependent on axonal transport through sensory neurons, as evidenced by colchicine injections (5 mM, 2 µl) into the trigeminal ganglion completely preventing BoNT/A effects. ----- CONCLUSIONS/SIGNIFICANCE: Two different types of pain, IoNC and formalin, are accompanied by dural extravasation. The lasting effect of a unilateral injection of BoNT/A in experimental animals suggests that BoNT/A might have a long-term beneficial effect in craniofacial pain associated with dural neurogenic inflammation. Bilateral effects of BoNT/A and dependence on retrograde axonal transport suggest a central site of its action

Comparison of analgesic effects of single versus repeated injection of botulinum toxin in orofacial formalin test in rats

Long-term effectiveness and repeated administration of botulinum toxin A are the basis for its use in both neuromuscular disorders and certain painful conditions. Botulinum toxin A has been recently approved for migraine treatment, and its off-label use extends to other craniofacial pain disorders. However, recently it was reported that, after repeated injection, botulinum toxin loses its antinociceptive efficacy in rats. In present study with a similar design, we compared the effects of single and repeated injections of botulinum toxin in formalin-induced orofacial pain. No statistically significant differences were found between single or repeatedly treated animal groups. Our results are in line with the clinical experience and suggest that botulinum toxin can be re-administered in orofacial pain treatment

Botulinum toxin A, brain and pain

Botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins known and a potential biological threat. At the same time, it is among the most widely used therapeutic proteins used yearly by millions of people, especially for cosmetic purposes. Currently, its clinical use in certain types of pain is increasing, and its long-term duration of effects represents a special clinical value. Efficacy of BoNT/A in different types of pain has been found in numerous clinical trials and case reports, as well as in animal pain models. However, sites and mechanisms of BoNT/A actions involved in nociception are a matter of controversy. In analogy with well known neuroparalytic effects in peripheral cholinergic synapses, presently dominant opinion is that BoNT/A exerts pain reduction by inhibiting peripheral neurotransmitter/inflammatory mediator release from sensory nerves. On the other hand, growing number of behavioral and immunohistochemical studies demonstrated the requirement of axonal transport for BoNT/A's antinociceptive action. In addition, toxin's enzymatic activity in central sensory regions was clearly identified after its peripheral application. Apart from general pharmacology, this review summarizes the clinical and experimental evidence for BoNT/A antinociceptive activity and compares the data in favor of peripheral vs. central site and mechanism of action. Based on literature review and published results from our laboratory we propose that the hypothesis of peripheral site of BoNT/A action is not sufficient to explain the experimental data collected up to now

Evidence for central antispastic effect of botulinum toxin type

Background and Purpose Botulinum toxin type A (BoNT/A) injections into hyperactive muscles provide effective treatment for spasticity and dystonias, presumably due to its local effects on extrafusal and intrafusal motor fibres. A recent discovery of toxin's retrograde axonal transport to CNS might suggest additional action sites. However, in comparison to cholinergic peripheral terminals, functional consequences of BoNT/A direct central action on abnormally increased muscle tone are presently unknown. To address this question, the central effects of BoNT/A were assessed in experimental local spastic paralysis. ----- Experimental Approach Local spastic paralysis was induced by injection of tetanus toxin (1.5 ng) into rat gastrocnemius. Subsequently, BoNT/A (5 U·kg−1) was applied i.m. into the spastic muscle or intraneurally (i.n.) into the sciatic nerve to mimic the action of axonally transported toxin. Functional role of BoNT/A transcytosis in spinal cord was evaluated by lumbar i.t. application of BoNT/A‐neutralizing antitoxin. BoNT/A effects were studied by behavioural motor assessment and cleaved synaptosomal‐associated protein 25 (SNAP‐25) immunohistochemistry. ----- Key Results Tetanus toxin evoked muscular spasm (sustained rigid hind paw extension and resistance to passive ankle flexion). Subsequent injections of BoNT/A, i.m. or i.n, reduced tetanus toxin‐evoked spastic paralysis. Beneficial effects of i.n. BoNT/A and occurrence of cleaved SNAP‐25 in ventral horn were prevented by i.t. antitoxin. ----- Conclusions and Implications Axonally transported BoNT/A relieves muscle hypertonia induced by tetanus toxin, following the trans‐synaptic movement of BoNT/A in the CNS. These results suggest that such direct, centrally mediated reduction of abnormal muscle tone might contribute to the effectiveness of BoNT/A in spasticity and hyperkinetic movement disorders

Središnje antinociceptivno djelovanje botulinum toksina A

Background: Botulinum toxin type A (BoNT/A) is an emerging long-acting therapeutic for chronic pain. In contrast to previously assumed local action, novel evidence point to the CNS as the possible site of BoNT/A action on pain after its axonal transport. The aim of this thesis was to characterize the sites and mechanisms of BoNT/A action on central pain transmission. ----- Methods: BoNT/A antinociceptive activity was characterized by behavioral nociceptive assessment, immunodetection of BoNT/A enzymatic product (cleaved synaptosomal-associated protein 25 (SNAP-25)) and c-Fos neuronal activation in different rat sensory regions. Peripheral, intraneural and intraganglionic BoNT/A injections, and microtubule-blocker colchicine were employed to assess BoNT/A axonal transport in peripheral sensory nerves. By employing trigeminal nerve ablation we examined possible transcytosis of BoNT/A in sensory regions. Denervation of trigeminal afferents with capsaicin was employed to examine the potential role of capsaicin-sensitive (vanilloid 1-expressing) neurons. ----- Results: Microtubule-dependent axonal transport of BoNT/A, necessary for its antinociceptive activity, occurred in sensory neurons. Following different toxin peripheral injections, cleaved SNAP-25 has been observed in corresponding sensory nuclei of brainstem (trigeminal nucleus caudalis) and spinal cord dorsal horn, but not in higher level sensory areas. BoNT/A enzyme activity was localized presynaptically in capsaicin-sensitive (vanilloid 1 receptor -expressing) central afferent terminals. BoNT/A reduced the pain-associated neuronal activation in TNC and supramedullary regions involved in descending pain control. ----- Conclusion: After its axonal transport in sensory neurons, BoNT/A modulates pain transmission at the central synapse of primary afferents. Involvement of capsaicin-sensitive neurons is associated with the selectivity of BoNT/A action for pain hypersensitivity. These findings contribute to the explanation of BoNT/A mechanisms of action in pain and possible refinement of its clinical use.UVOD I CILJ ISTRAŽIVANJA: Botulinum toksin tipa A (BoNT/A), neurotoksin iz anaerobne bakterije Clostridium botulinum, je jedan od najpotentnijih bioloških toksina. Ulaskom u živčane terminale uzrokuje enzimsko cijepanje sinaptosomalnog proteina molekulske mase od 25 kDa (eng. synaptosomal-associated protein of 25 kDa; SNAP-25), što sprječava lučenje neurotransmitora. Intoksikacija organizma preko hrane ili infekcija sporama bakterija pri određenim uvjetima uzrokuje neuroparalitičku bolest botulizam koju karakterizira kljenut mišića. Pročišćeni farmakološki pripravak u malim dozama se koristi kao terapija određenih hiperkinetskih poremećaja pokreta i autonomnih poremećaja, te kao kozmetički pripravak za smanjenje bora. Zbog dugotrajnog djelovanja nakon jednokratne primjene koji traje nekoliko mjeseci BoNT/A se sve više koristi u liječenju određenih tipova kronične boli. BoNT/A je odobren za liječenje kronične migrene, a njegova učinkovitost je pokazana kod niza drugih bolnih poremećaja, poput različitih vrsta neuropatskih boli, artritisa, boli u leđima, temporomandibularnih poremećaja, miofascijalne boli, itd. Zajedničko obilježje svih navedenih poremećaja je postojanje bolne preosjetljivosti i centralne senzitizacije. Posebne prednosti njegove primjene, osim dugotrajnog djelovanja, su manje nuspojave nego kod konvencionalnih analgetika poput opioida, te manjak utjecaja na transmisiju akutne nociceptivne boli. Unatoč sve značajnijoj kliničkoj uporabi, malo se zna o mjestu i mehanizmu antinociceptivnog djelovanja BoNT/A. Prevladavajuće hipoteze predložene od strane proizvođača farmakološkog pripravka su da BoNT/A sprječava lokalno lučenje neurotransmitora glutamata i neuropeptidnih posrednika upale s perifernih senzornih završetaka, te na taj način inhibira perifernu transmisiju boli. Novi eksperimenti, poglavito iz našeg laboratorija, upućuju na manjak povezanosti perifernog učinka BoNT/A s njegovim antinociceptivnim djelovanjem. U bilateralnim modelima boli, BoNT/A je nakon jednostrane primjene pokazao bilateralan antinociceptivni učinak, ovisan o aksonalnom transportu kroz periferne neurone. Ti eksperimenti, potvrđeni na nekoliko eksperimentalnih modela, su ukazali na moguće centralno mjesto djelovanja BoNT/A posredovano aksonalnim transportom. Stoga, cilj ovog doktorata je bilo ispitati mogući aksonalni transport molekula BoNT/A u središnji živčani sustav kroz senzorne neurone, okarakterizirati mjesta njegova djelovanja na centralnu transmisiju boli, te ispitati mehanizam selektivnosti djelovanja na bolnu preosjetljivost u odnosu na akutnu senzornu transmisiju. ----- METODE: Moguće djelovanje enzimski aktivnog BoNT/A u centralnim senzornim regijama štakora je istraženo imunodetekcijom enzimskog produkta djelovanja BoNT/A (pocijepani odn. krnji SNAP-25) nakon različitih načina periferne primjene: subkutanog davanja u područje trigeminusa (područje brkova na licu) ili u stražnju šapicu, te nakon injiciranja u mišić gastrocnemius ili nakon direktne injekcije u izolirani ishijadikus. Paralelno s mikroinjiciranjem u ishijadikus primijenili smo i kolhicin, kako bismo potvrdili mogući aktivni mikrotubularni mehanizam aksonalnog transporta kroz periferne živce. Da bismo potvrdili ulogu aksonalnog transporta BoNT/A kroz senzorne neurone, antinociceptivno djelovanje BoNT/A je ispitano u modelu orofacijalne boli uzrokovane formalinom nakon periferne i(ili) intraganglijske primjene BoNT/A, i intraganglijskog kolhicina. Nakon davanja toksina u područje lica, imunohistokemijskom lokalizacijom pocijepanog SNAP-25 smo pokušali utvrdili mjesto djelovanja BoNT/A u središnjem živčanom sustavu. Pojava pocijepanog SNAP-25, osim u kaudalnoj jezgri trigeminusa, je ispitana i u supraspinalnim senzornim regijama poput talamusa, moždane kore, hipotalamusa, locus coeruleus-a itd. Ablacijom trigeminalnog živca smo provjerili mogućnost transcitoze BoNT/A u kaudalnoj jezgri trigeminusa. Pokusima u kojima smo primijenili konfokalnu mikroskopiju smo provjerili kolokalizaciju pocijepanog SNAP-25 s neuronskim i staničnim markerima sinapsi, aksona, dendrita, neuronskih jezgri, astrocita itd. Kako bismo dodatno istražili regionalna mjesta djelovanja BoNT/A na centralnu transmisiju boli, istražili smo neuronsku aktivaciju u trigeminalnoj jezgri i drugim supraspinalnim nociceptivnim regijama imunohistokemijskom analizom ekspresije c-Fos proteina nakon bolnog podražaja uzrokovanog formalinom. Budući da BoNT/A ne djeluje na akutnu transmisiju boli i ostalih senzornih podražaja, ispitali smo moguću povezanost njegova antinociceptivnog djelovanja s neuronima osjetljivim na kapsaicin. Nakon injekcije BoNT/A u trigeminalnu regiju, istražili smo pojavnost pocijepanog SNAP-25 u kapsaicin-osjetljivim neuronima kolokalizacijom pocijepanog SNAP-25 s vaniloidnim-1 receptorom prolaznog receptorskog potencijala (TRPV1), te denervacijom trigeminalnih aferentnih neurona s intraganglijskom injekcijom visoke doze kapsaicina (2%). Denervacijskim postupkom također smo istražili ulogu kapsaicin-osjetljivih nociceptora pri antinocicepcijskom djelovanju BoNT/A u modelu orofacijalne boli uzrokovane formalinom. Dalje, ispitali smo moguću povezanost djelovanja BoNT/A i neuropeptida povezanog s genom za kalcitonin (eng. calcitonin gene-related peptide, CGRP), koji posreduje središnju senzitizaciju kod boli i migrene. Mogući utjecaj BoNT/A na lučenje i ekspresiju CGRP-a smo istražili imunodetekcijskim mjerenjem koncentracije CGRP-a u cerebrospinalnom likvoru pri različitim bolnim podražajima metodama enzimske imunoadsorpcijske analize (ELISA) i radioimunološkog testa. Nadalje, ispitali smo kolokalizaciju CGRP-a i pocijepanog SNAP-25 u trigeminalnoj jezgri i kranijalnoj duri, te utjecaj perifernog BoNTA na ekspresiju CGRP-a u gangliju, kranijalnoj duri i trigeminalnoj jezgri. ----- REZULTATI: Pocijepani SNAP-25 se nakon različitih mjesta periferne primjene BoNT/A pojavio u odgovarajućim senzornim nociceptivnim regijama (trigeminalna kaudalna jezgra i stražnji rog leđne moždine. Također, pocijepani SNAP-25 se pojavio i u prednjem rogu leđne moždine. Pojava pocijepanog SNAP-25 u leđnoj moždini nakon primjene BoNT/A u periferni živac je spriječena blokadom aksonalnog transporta pomoću kolhicina. BoNT/A primijenjen periferno i intraganglijski je smanjio bolnu preosjetljivost kod štakora u drugoj fazi formalinskog testa. Kolhicin primijenjen intraganglijski spriječio je antinociceptivno djelovanje BoNT/A na drugu fazu orofacijalnog formalinskog testa. Nakon ablacije trigeminalnog živca došlo je do unilateralne osjetne denervacije, ali i nestanka imunoreaktivnosti pocijepanog SNAP-25 u kaudalnoj jezgri trigeminusa, što ukazuje na enzimsku aktivnost BoNT/A u središnjim završecima osjetnih neurona. Kolokalizacija pocijepanog SNAP-25 je pokazala da je enzimski aktivni BoNT/A bio prisutan u središnjim sinapsama i aksonima. Pocijepani SNAP-25 nakon periferne primjene BoNT/A nije bio vidljiv u supraspinalnim regijama. BoNT/A je smanjio nociceptivnu aktivaciju neurona (mjerenu pomoću ekspresije c-Fos proteina) u kaudalnoj jezgri trigeminusa, locusu coeruleusu i periakveduktalnoj sivoj tvari. Pocijepani SNAP-25 se nakon facijalne primjene pojavio u središnjim živčanim završecima neurona osjetljivih na kapsaicin koji izražavaju TRPV1 receptor. Učinak BoNT/A na orofacijalnu bol uzrokovanu formalinom, te pojava pocijepanog SNAP-25 u CNS-u su spriječeni denervacijom neurona osjetljivih na kapsaicin. Mjerenjem koncentracije CGRP-a u cerebrospinalnom likvoru nije bilo moguće utvrditi utjecaj BoNT/A na središnje lučenje neuropeptida. Kod eksperimentalne boli uzrokovane injekcijom Freundovog adjuvansa u područje čeljusnog zgloba, BoNT/A je značajno spriječio pojačanu ekspresiju CGRP-a u kranijalnoj duri. U trigeminalnoj jezgri nije bilo značajne kolokalizacije pocijepanog SNAP-25 i CGRP-a. dok se pocijepani SNAP-25 u kranijalnoj duri pojavio u aferentima koji sadrže CGRP. ----- ZAKLJUČCI: Aksonalni transport enzimski aktivnog BoNT/A kroz senzorne neurone posredovan mikrotubulima odgovoran je za njegovo djelovanje na bol. Antinociceptivni učinak BoNT/A je povezan s njegovom enzimskom aktivnošću na centralnim završecima senzornih neurona osjetljivih na kapsaicin. Djelovanje BoNT/A na bol i centralnu senzitizaciju je praćeno smanjenjem aktivacije neurona u dorzalnom rogu i nekim supramedularnim regijama uključenima u silaznu inhibiciju boli. Selektivna uključenost neurona osjetljivih na kapsaicin objašnjava i selektivnost djelovanja BoNT/A na alodiniju i hiperalgeziju kod određenih kroničnih bolnih stanja, te manjak učinka na normalni prijenos akutne nociceptivne boli. Nalaz da BoNT/A smanjuje neurotransmisiju CGRP-a u kranijalnoj duri mogao bi objasniti njegov učinak na kroničnu migrenu. Ovi nalazi doprinose razumijevanju djelovanja BoNT/A na bolne poremećaje i mogućem poboljšanju njegove kliničke primjene