Capsaicin protects neuromuscular junctions from the inhibitory effects of botulinum neurotoxin A

Within 24 hrs after injecting botulinum neurotoxin A (BoNT/A) into the hindlimb, mice lost the toe spread reflex and developed progressive muscle weakness. At the same time, the compound muscle action potential amplitude decreased. Injection of capsaicin before BoNT/A significantly reduced these affects and protected the muscle twitch tension of the Extensor digitorum longus (EDL) nerve muscle preparation. Acute in vitro exposure of isolated nerve muscle preparations, as well as Neuro 2a cells, to capsaicin prevented uptake of Alexa 647 BoNT/A. Motor nerve endings as well as Neuro 2a cells express the capsaicin receptor, a transient receptor potential channel of the vanilloid family (TRPV1). Capsaicin as well as disruption of clathrin coated pits (CCPs) reduced Neuro 2a cell uptake of BoNT/A. FM1-43 uptake indicated that exocytosis persists for BoNT/A treated Neuro 2a cells pretreated with capsaicin. Pre-injection of wortmannin (WMN), a PI3Kinase inhibitor, also protected mice from the paralytic effects of BoNT/A. When applied alone, either WMN or capsaicin selectively reduced stimulus-evoked transmitter release from motor nerve endings. We hypothesize that TRPV1 activation reduces PI(4,5)P2 level within the membrane. This prevents CCP formation and uptake of BoNT/A

Botulinum Toxin as Preventive Treatment for Migraine: A Randomized Double-Blind Study

Aim: To determine if botulinum toxin type A (BoNT-A) injections can reduce the frequency and severity of migraines. Methods: Patients (n = 127) were randomized to receive placebo or two doses of BoNT-A (Dysport (R)). The primary endpoint was reduction in number of migraine attacks up to week 8 and between weeks 8 and 12 after injection. Patient diaries were used to record secondary endpoints, including frequency, severity and duration of migraine attacks. Results: There was a mean reduction of 0.54 and 0.94 attacks/month with placebo and BoNT-A, respectively, and absolute attack count was less in the verum group (3.6 vs. 4.2 attacks/month), but this was not statistically significant. The patients' global assessment of efficacy was significantly better than placebo in the high-dose group (p = 0.02) but no effects were seen for the other secondary efficacy parameters. Conclusion: Our study showed a trend towards a reduced attack rate with verum but did not show any statistically significant efficacy of BoNT-A in the prophylactic treatment of migraine. Copyright (C) 2009 S. Karger AG, Base

SNARE based peptide linking as an efficient strategy to retarget botulinum neurotoxin’s enzymatic domain to specific neurons using diverse neuropeptides as targeting domains

Many disease states are caused by miss-regulated neurotransmission. A small fraction of these diseases can currently be treated with botulinum neurotoxin type A (BoNT/A). BoNT/A is composed of three functional domains – the light chain (Lc) is a zinc metalloprotease that cleaves intracellular SNAP25 which inhibits exocytosis, the translocation domain (Td) that enables the export of the light chain from the endosome to the cytosol, and the receptor binding domain (Rbd) that binds to extracellular gangliosides and synaptic vesicle glycoproteins while awaiting internalisation [1]. Current endeavours are directed towards retargeting Bont/A as well as finding safer methods of preparation and administration. Recently, our laboratory has developed a SNARE based linking strategy to recombine non-toxic BoNT/A fragments into a functional protein by simple mixing [2]. This SNARE based linking strategy permits the stepwise assembly of highly stable macromolecular complexes [2,3]. Onto these three SNARE peptides, diverse functional groups can be attached to the N- or C- terminus by direct synthesis and/or by genetic design. To enhance the therapeutic potential of BoNT/A, this method enables the rapid assembly of a large array of neuropeptide-SNAREs to their cognate LcTd-SNARE. A substitution of the Rbd with various neuropeptide sequences permits a large throughput combinatorial assay of LcTd to target new cell types. In this study, we have fused LcTd to 3 different Synaptobrevin sequences; we also use a small protein staple, and 26 different Syntaxin-neuropeptide fusions (permitting the assay of 78 new chimeric LcTd proteins with modified targeting domains). These neuropeptides such as, but not exclusively, somatostatin (SS), vasoactive intestinal peptide, substance P, opioid peptide analogues, Gonadotropin releasing hormone, and Arginine Vasopressin, which natively function through G protein coupled receptors (GPCR) can undergo agonist induced internalisation upon activation. The ability of our new constructs, once endocytosed, to inhibit neurotransmitter release was tested on different neuronal cell lines with immunoblotting of endogenous SNAP25. This cleavage by Lc reflects the ultimate readout of the enzyme’s efficacy, which incorporates the cell surface binding, internalisation kinetics, translocation of the Lc to the cytosol, and finally the enzymatic cleavage of SNAP25. Internalisation of the toxins can also be monitored with confocal microscopy and FACS by the substitution of the staple peptide for a fluorescent homologue. Figure 1 shows that whole boNT/A (upper left) can have its Rbd replaced with SNARE peptides, which will fuse together to form highly stable chimeric proteins with an altered targeting domain (right). Figure 1 also shows 4 different neuropeptide synthaxins in complex, resolved on SDS-PAGE gel (bottom left lanes 1-4, boiled 1’-4’). Fig. 1. SNARE-linked botulinum neurotoxins used for the retargeting of Bont/A. 29

A Targeted Therapeutic Rescues Botulinum Toxin-A Poisoned Neurons

Botulinum neurotoxin (BoNT), a Category A biothreat agent, is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. BoNT acts specifically by blocking neurotransmission primarily at peripheral nerve-muscle junctions causing severe flaccid muscle paralysis, which is fatal if proper medical care is not provided. The neurotoxin acts by specifically entering the presynaptic nerve endings where it interferes with the biochemical machinery involved in the process of neurotransmitter release, i.e., neuroexocytosis. Most serotypes of BoNT are known to remain active for weeks to months after entering the nerves, but BoNT/A is the most potent and long lasting in causing muscle paralysis. An effective medical countermeasure strategy requires developing a drug that could rescue poisoned neuromuscular synapses, and would include its efficient delivery specifically to presynaptic nerve terminals. Here we report rescuing of botulinum poisoned nerve cells by Mastoparan-7 (Mas-7), a peptide constituent of bee venom, that was delivered through a drug delivery vehicle (DDV) constructed from the non-toxic fragment of botulinum neurotoxin itself. We found that Mas-7 that was delivered into BoNT/A intoxicated cultured mouse spinal cord cells restored over 40% of stimulated neurotransmitter release. The rescue of the cell poisoning did not occur from inhibition of the endopeptidase activity of BoNT/A against its well known substrate, SNAP-25 that is mechanistically involved in the exocytosis process. Rather, Mas-7 induced a physiological host response apparently unrelated to SNAP-25, but linked to the phospholipase signal transduction pathway. In addition to providing the first effective antidote against botulism, our results open new avenues to study the mechanism of exocytosis, and also to examine an alternative cellular mechanism of botulinum neurotoxin action. An effective BoNT-based DDV can also be utilized for drug delivery against many neuronal and neuromuscular disorders

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

Safety and efficacy of a propofol and ketamine based procedural sedation protocol in children with cerebral palsy undergoing botulinum toxin A injections.

Background Pediatric patients with cerebral palsy (CP) often undergo intramuscular botulinum toxin (BoNT‐A) injections. These injections can be painful and may require procedural sedation. An ideal sedation protocol has yet to be elucidated. Objective To investigate the safety and efficacy of a propofol and ketamine based sedation protocol in pediatric patients with cerebral palsy requiring BoNT‐A injections. Design This is a retrospective chart review of children with CP undergoing propofol and ketamine based sedation for injections with botulinum toxin A. Setting The sedations took place in a procedural sedation suite at a tertiary children’s hospital from Feb 2013 through Sept 2017. Patients 164 patients with diagnoses of cerebral palsy were included in this study. Methods An initial bolus of 0.5 mg/kg ketamine followed by a 2 mg/kg bolus of propofol was administered with supplemental boluses of propofol as needed to achieve deep sedation during the intramuscular BoNT‐A injections. Main Outcome Measurements Propofol dosages, adverse events, serious adverse events, and sedation time parameters were reviewed. Results 345 sedations were successfully performed on 164 patients. The median total dose of propofol was 4.7 mg/kg (IQR 3.5, 6.3). Adverse events were encountered in 10.1% of procedures including hypoxemia responsive to supplemental oxygen (9.6%) and transient apnea (1.4%). The mean procedure time, recovery time and total sedation time were 10, 11 and 33 minutes, respectively. With regard to patient variables, including age, weight, dose of propofol, sedation time, and Gross Motor Function Classification System classification, there was no association with increased incidence of adverse events. Conclusion Our sedation protocol of propofol and ketamine is safe and effective in children with cerebral palsy undergoing procedural sedation for intramuscular injections with BoNT‐A. The adverse events encountered appeared to be related to airway and respiratory complications secondary to musculoskeletal deformities, emphasizing the importance of airway monitoring and management in these patients

片側末梢投与されたA型ボツリヌス毒素は動物モデルにおいて両側三叉神経節に局在する

Peripheral nerve injury leads to sensory ganglion hyperexcitation, which increases neurotransmitter release and neuropathic pain. Botulinum toxin type A (BoNT/A) regulates pain transmission by reducing neurotransmitter release, thereby attenuating neuropathic pain. Despite multiple studies on the use of BoNT/A for managing neuropathic pain in the orofacial region, its exact mechanism of transport remains unclear. In this study, we investigated the effects of BoNT/A in managing neuropathic pain in two different animal models and its transport mechanism in the trigeminal nerve. Intraperitoneal administration of cisplatin induced bilateral neuropathic pain in the orofacial region, reducing the head withdrawal threshold to mechanical stimulation. Unilateral infraorbital nerve constriction (IONC) also reduced the ipsilateral head withdrawal threshold to mechanical stimulation. Unilateral peripheral administration of BoNT/A to the rat whisker pad attenuated cisplatin-induced pain behavior bilaterally. Furthermore, contralateral peripheral administration of BoNT/A attenuated neuropathy-induced behavior caused by IONC. We also noted the presence of BoNT/A in the blood using the mouse bioassay. In addition, the Alexa Fluor-488-labeled C-terminal half of the heavy chain of BoNT/A (BoNT/A-Hc) was localized in the neurons of the bilateral trigeminal ganglia following its unilateral administration. These findings suggest that axonal and hematogenous transport are involved in the therapeutic effects of peripherally administered BoNT/A in the orofacial region

Efficacy of Botulinum Toxin Type A Injection Compared to Rehabilitation in the Treatment of Upper Limb Post-Stroke Spasticity

Abstract Introduction: The standard treatment for post-stroke spasticity (PSS) is rehabilitation. Botulinum Toxin Type A (BoNT-A) injection is used in treatment for PSS. This review analyzes the efficacy of BoNT-A injection on arm muscle tone and function compared with rehabilitation in adult patients with upper limb spasticity after stroke. Methods: A literature search was conducted through PubMed Clinical Queries and ScienceDirect in November 2019. 7 articles consisting of randomized clinical trials were selected based on set criteria. Results: Five studies found BoNT-A had a significant difference reducing muscle tone compared to rehabilitation alone. Three studies found a significant functional improvement in the limb with the BoNT-A injection group using primary outcome measures. However, one of these compared the BoNT-A/rehabilitation group for functional improvement in the limb to BoNT-A/no rehabilitation group, suggesting the rehabilitation intervention improved the function. Discussion: Most of the studies supported that BoNT-A can reduce muscle tone more significantly rehabilitation alone. The studies lacked consistent data that BoNT-A, when supplemented to rehabilitation, shows a significant functional improvement than rehabilitation alone. Study limitations included: blindness, funding bias, smaller sample sizes and unclear rehabilitation protocols. Conclusion: Patients treated with BoNT-A and rehabilitation can have a reduction in spastic muscle tone but not an overall improved function of the limb as compared to rehabilitation alone. The results support rehabilitation as mainstay of treatment. BoNT-A should remain available to patients as adjuvant treatment. Current research needs to be expanded with larger sample sizes, standardized dosing schedule, and defined rehabilitation protocols

Control of Autophagosome Axonal Retrograde Flux by Presynaptic Activity Unveiled Using Botulinum Neurotoxin Type A

Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity