Botulinum Neurotoxin Research Articles

Botulinum neurotoxins (BoNTs) are the bacterial proteins responsible for the flaccid paralysis and lethal effects of botulism. They act by inhibiting neurotransmitter release, primarily at peripheral cholinergic nerve terminals, and have traditionally been classified into seven serotypes, designated with letters A through G. Over a long history of investigation, scientists and physicians have learned to harness the selectivity and potency of these neurotoxins to advance therapeutic applications. These advances have come by leveraging the basic science understanding of how the BoNT's work. The BoNTs are typically produced as inactive single-chain proteins of 150kDa that can be proteolytically activated to form a 50kDa light chain (LC) and 100kDa heavy chain (HC) that remain linked by a disulfide bond. The BoNTs act through a multi-step mechanism in which the HC mediates receptor binding and translocation of the LC into the neuronal cell cytosol. The LC is a zinc endopeptidase and each BoNT serotype cleaves a unique and specific bond within the three-protein soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. In the late 1990s, I had the opportunity to help visualize the atomic structure of this formidable molecule, a story I share to honor the legacy of Hans Bigalke, whose work set the stage for new careers and discoveries in BoNT research and medicine.

Neuroinflammation and oxidative stress are key drivers of various ocular diseases. Experimental hypoxia, modeled using cobalt chloride (CoCl2), induces hypoxia-inducible factor 1-alpha (HIF-1α) stabilization, mitochondrial dysfunction, and excessive reactive oxygen species (ROS) production, primarily via the NADPH oxidase 2 (Nox2)–voltage-gated proton channel Hv1 axis. Although Botulinum neurotoxin type A (BoNT/A) is classically recognized for SNAP-25 cleavage, recent studies suggest broader anti-inflammatory and neuroprotective effects. We evaluated BoNT/A in R28 retinal precursor cells and ex vivo retinal explants subjected to CoCl2-induced hypoxic stress. BoNT/A pretreatment attenuated CoCl2-induced upregulation of HIF-1α, Hv1, Nox2, NOD-like receptor protein 3 (NLRP3), COX2, and nuclear factor kappa B (NF-κB), while enhancing protective mediators including suppressor of cytokine signaling 3 (SOCS3), Growth Associated Protein 43 (Gap43), and Syntaxin12. Brn3a expression and retinal architecture were preserved, apoptotic cell death reduced, and glial activation suppressed. Moreover, BoNT/A decreased mitochondrial ROS accumulation, restored voltage-dependent anion channel 1 (VDAC1) distribution, and partially stabilized intracellular pH. These findings indicate that BoNT/A mitigates oxidative stress and inflammation in hypoxia-driven retinal injury, at least in part, via modulation of the Nox2–Hv1–ROS axis, and support its potential as a therapeutic candidate for ocular disorders associated with hypoxia and neuroinflammation.

Diabetic neuropathy is characterized by nerve damage and chronic neuropathic pain and lacks effective treatment. Botulinum neurotoxin type A (BoNT/A), a neurotoxin with established therapeutic use in neurological disorders, has emerged as a potential analgesic agent. This study investigated the effects of a recombinant form of BoNT/A1 (rBoNT/A1) on neuropathic pain induced by spared nerve injury (SNI) in a diabetic mouse model. Thirty-two adult male C57BL/6JRj diabetic mice were subjected to SNI or sham surgery. Fourteen days post surgery, mice received an intraplantar dose of rBoNT/A1 or vehicle. Mechanical allodynia was assessed using von Frey filaments, and spinal cord and sciatic nerve tissues were analyzed via immunohistochemistry and transmission electron microscopy to evaluate glial activation, neurotransmitter receptor expression, and axonal morphology. The results demonstrated that rBoNT/A1 significantly alleviated mechanical allodynia and caused a marked reduction in Iba1-positive microglial activation in the spinal cord, whereas no significant changes were observed in astrocyte (GFAP) density or GABAAR subunit expression. Additionally, rBoNT/A1 treatment did not significantly alter axon diameter, myelin thickness, or C-fiber morphology. In conclusion, intraplantar administration of rBoNT/A1 reduced SNI-induced mechanical allodynia in diabetic mice, potentially by attenuating spinal microglial activation, supporting the therapeutic promise of rBoNT/A1 in managing diabetic neuropathic pain.

Zebrafish in neurotoxin research: Insights into botulinum toxicity, mechanisms, and therapy.

In silico multiscale computational modelling of botulinum toxin a diffusion for glabellar wrinkle treatment: Optimizing injection volumes across formulations.

Is the use of botulinum toxin injection for treating neurological disorders safe during pregnancy and lactation?

Botulinum toxin treatment of lingual dystonia, oromandibular dystonia and bruxism-A review and update.

Three-year outcomes of repeated botulinum neurotoxin A injections to the lower extremities in young children with spastic cerebral palsy in GMFCS levels I to III.

ABSTRACT In the ancient Roman world, cattle played an integral role in daily agricultural tasks, providing the means necessary to plow fields, mill grains, and transport goods. The research presented here deals with the remains of 14 cattle discovered in a mass grave at the Roman villa of Vilauba in Catalonia, Spain. According to the archeological record, it can be ruled out that the animals were slaughtered for consumption, banqueting, or sacrificial purposes. By investigating the metagenomic sequences generated from the bovine remains, we identified in three individuals a group I Clostridium strain, phylogenetically related to known producers of botulinum neurotoxins – suggesting that the Vilauba strain may have had toxigenic potential. Moreover, we discovered a Mycolicibacterium species phylogenetically related to known opportunistic pathogens. While no definitive conclusions can be drawn about disease, the phylogenetic placement of these taxa and the detection of Clostridium virulence-associated genes suggest a possible role beyond postmortem contamination. Collectively, these findings draw attention to atypical bacterial species, such as C. sporogenes, which are often overlooked in palaeogenomic studies due to their ambiguous status as environmental microbes, commensals, or potential pathogens. Their detection in animal remains highlights that they may represent a blind spot in our current understanding of livestock health. More broadly, this study underscores the current complexity of investigating such taxa and emphasizes the need for novel methods to disentangle the roles of these bacterial species.

Botulinum neurotoxin A (BoNT/A) is one of the most toxic substances known. The bacterium Clostridium botulinum produces it as a 150-kDa polypeptide with relatively low potency. Surprisingly, the endogenous protease that transforms this polypeptide into an activated, full-potency toxin, consisting of a 100-kDa heavy chain connected to a 50-kDa light chain (LC) by a disulfide bond, is still unknown. This study aimed to identify the BoNT/A-activating protease. We screened cation-exchange chromatography fractions of C. botulinum A culture supernatant for activity using a toxin-simulating substrate comprising the LC and the translocation domain (HN). Proteomic analysis of the active fraction identified α-clostripain as a candidate BoNT/A-activating protease. Recombinant α-clostripain cleaved the simulating substrate between the toxin LC and HN. However, incubation of recombinant α-clostripain with recombinant inactivated BoNT/A (rBoNT/Ai) resulted in non-specific digestion of the toxin. Since similar non-specific digestion was observed also by C. botulinum A culture supernatant, we hypothesized that the toxin should be protected by an accessory protein to prevent non-specific cleavage. Indeed, incubation of rBoNT/Ai with α-clostripain or culture supernatant in the presence of recombinant NTNH (non-toxic non-hemagglutinin) resulted in specific cleavage of the toxin into 100- and 50-kDa fragments. Subsequently, we evaluated the activation of rBoNT/A by α-clostripain in a mouse model. Cleavage of rBoNT/A by α-clostripain in the presence of NTNH resulted in a 77-fold increase in toxicity, corresponding to toxin activation. To the best of our knowledge, these results elucidate the mechanism of BoNT/A activation for the first time.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-21372-0.

Disclosure: P. Roque: None. M. Lopez: None. T. Sanchez: None. M. Albano: None. P. Aquino: None. A. Lo: None.INTRO: Subacute thyroiditis resulting from locally injected Botox to the neck muscles is a rare complication from a seemingly straightforward aesthetic procedure. CASE: A 37-year-old female without prior thyroid disease presented with a 2-week history of palpitations, tremors, heat intolerance, and cold sweats following a noninvasive platysmaplasty via Botox injections. She had previously undergone said procedure without complications, however, her most recent treatment triggered said conditions. On examination, she was clinically thyrotoxic with direct tenderness over the thyroid gland. Initial tests were consistent with hyperthyroidism: TSH <0.07, FT4 2.3 and FT3 7.0, with ESR levels of 70 and negative TPO, TSI, and TRab receptor antibodies. Low uptake on RAI scan confirmed thyroiditis. She was prescribed NSAIDs and atenolol, and started on a 5-week tapering course of prednisone, leading to resolution of symptoms and normalization of thyroid tests. DISCUSSION: Subacute thyroiditis can be due to radiation, trauma, or infections; this case describes a rare instance of subacute thyroiditis following Botox injection into the platysmal bands. There is limited literature on the direct and indirect effects of Botox on the thyroid. Botox has been reported to trigger an autoimmune response in patients with underlying Hashimoto’s disease possibly from molecular mimicry. A study by Gregoric et al. found that eyelid injections of Botox on a Hashimoto patient caused persistent TSH elevation despite adequate levothyroxine therapy, likely due to anti-Botox antibodies inducing anti-TSH-R antibodies inhibiting TSH-R signaling. In this particular case, her lack of autoantibodies and unremarkable thyroid history argue against an autoimmune condition. The patient's favorable response to prednisone further supports the inflammatory nature of the thyroid dysfunction. Localized injections near the thyroid can lead to thyroid dysfunction through inadvertent thyroid manipulation or spillage of Botox into surrounding tissues, possibly causing an inflammatory response. In this case, absence of other triggers supports Botox as the primary cause of the thyroiditis. CONCLUSION: Botox injections to the cervical area used for both aesthetic and medical purposes are generally considered benign, however, can rarely trigger a severe thyroiditis reaction. Patients should be informed of said risk and monitor for possible symptoms of thyrotoxicosis. Early recognition of subacute thyroiditis allows for prompt diagnosis and appropriate management.Reference: Gregoric E, Gregoric JA, Guarneri F, Benvenga S. Injections of Clostridium botulinum neurotoxin A may cause thyroid complications in predisposed persons based on molecular mimicry with thyroid autoantigens. Endocrine. 2011 Feb;39(1):41-7. doi: 10.1007/s12020-010-9410-9. PMID: 21061092Presentation: Monday, July 14, 2025

IntroductionBenign essential blepharospasm (BEB) is a focal dystonia that can lead to functional blindness in older adults. While botulinum neurotoxin (BoNT) is widely utilized in the treatment of BEB, there remains a lack of consensus regarding the optimal injection technique. This study aims to compare the clinical outcomes and measurable scales associated with injections into the orbicularis oculi muscle at the pars pretarsalis (PPT) and pars preseptalis (PPS) regions, assessing their efficacy and side effects.MethodsThe study included 32 patients diagnosed with BEB. Two injection techniques utilizing onabotulinum toxin type-A—PPT and PPS—were compared. The modified Jankovic Scale (mJS) was used to assess the clinical severity and frequency of blepharospasm. At the same time, the Blepharospasm Disability Scale evaluated the associated disabilities. The Schirmer I test was used to assess dry eye symptoms post-BoNT administration. Adverse effects were self-reported, and participants were examined at 1- and 3-month follow-up.ResultsBoth injection techniques resulted in significant improvements in the mJS severity and frequency scores, as well as the Blepharospasm Disability Scale (p &amp;lt; 0.05). Notably, improvements in ocular dryness were particularly pronounced at the 1-month mark (p &amp;lt; 0.05). The PPT injection technique displayed a superior safety profile, with the total number of adverse events statistically greater in the PPS group compared to the PPT group (p &amp;lt; 0.05).ConclusionBoNT injections through both PPT and PPS techniques are effective in treating BEB in older adults. However, the PPT technique demonstrates a lower incidence of adverse events, rendering it a preferable choice for clinicians managing BEB.

Botulinum Neurotoxin Serotype A (BoNT/A), responsible for most human botulism cases, inhibits neurotransmitter release by cleaving the target protein SNAP-25. Previous literature demonstrated that BoNT/A mediated cleavage of a small subset of the SNAP-25 pool, resulting in SNAP-25 (1–197) fragments, is sufficient to block exocytosis. SNAP-25 (1–197) potentially competes against intact SNAP-25 for SNARE complexes and blocks neurotransmission through a dominant-negative mechanism. However, how a tiny fraction of cleaved SNAP-25 efficiently outcompetes a large pool of intact SNAP-25 remains unknown. Here, we examined the importance of SNAP-25 phosphorylation at Ser187 residue, located in the C-terminus SNARE domain, in the context of BoNT action. Our results demonstrated that Ser187-phosphorylated SNAP-25 can be efficiently cleaved in cells. Importantly, BoNT/A-cleaved SNAP-25 fragments in neuronal and non-neuronal cells are heavily phosphorylated at Ser187 and localized on the cell membrane. SNAP-25 (1–197) binds to syntaxin-1A, and the interaction is enhanced by Ser187 phosphorylation. We also found that SNAP-25 (1–197) survives longer than the BoNT/A enzymatic component itself in cells. Molecular modeling suggested that SNAP-25 (1–197), phosphorylated or not, forms stable SNARE complexes; however, Ser187 phosphorylation induces local changes in surface electrostatic potential and dynamics of the complex. This study characterizes the molecular mechanism underlying the dominant-negative effect of SNAP-25 (1–197) on neurotransmission. This research could have implications for the future development of BoNT/A inhibitors and the generation of new BoNT/A clinical formulations by regulating the abundance of Ser187 phosphorylation in cleaved SNAP-25 fragments.

Introduction: Botulinum neurotoxins are the most poisonous substances reported and listed in category ‘A’ of biowarfare agents. As serotype identification is a time-consuming process and there is no antidote commercially available, the development of inhibitors against serotypes causing human botulism would be beneficial. In the present study, a ligand-based in silico method was applied to identify the “hits” that could have the potential to act as countermeasures against human-intoxicating BoNTs. Methods: For this purpose, a computational approach using Molegro Virtual Docker and Auto- Dock tools was performed, where around thirty-five derivatives were designed and docked into the catalytic domain of BoNT/A, B, E, and F. The designed compounds were also studied for their ADME properties using an online web tool. Results and Discussion: Analysis of the molecular docking data of the complex by Molegro Virtual Docker revealed a high binding affinity between the target and designed ligands, with the MolDock score between -139.85 and -88.24 kcal/mol, whereas the AutoDock score ranged between -11.65 and -5.30 kcal/mol. Three SMNPIs, A11, A18, and A20, exhibited better binding affinities with the target proteins BoNT/A, /B, E, and /F and could be potential pan-active inhibitors. The ADME/T study showed that the designed ligands were less toxic and possessed drug-resemblance properties by considering the Lipinski, Ghose, Veber, and Egan rules, with a bioavailability score of 0.56. Conclusion: Our study provides insight into ‘hits’, which can lead to further progress in experimental studies and the development of new antidotes for botulism.

Essential tremor (ET) is a chronic, progressive condition classically affecting the hands and arms, is typically bilateral, and may be asymmetric. Tremor related disability can impact mood and quality of life. Disease progression and patient related factors may limit efficacy and use of traditional pharmacologic options. Botulinum neurotoxin (BoNT) injections have been explored as an alternative treatment option. This systematic review aims to evaluate the efficacy and safety of BoNT injections as a therapeutic intervention for essential tremors. Following PRISMA guidelines, a systematic literature search was conducted on PubMed, Cochrane, HERDIN, and ScienceDirect on March 2025. The following search string was used ("Essential tremor" OR "tremor disorder") AND ("botulinum toxin" OR "neurotoxin" OR "botulinum toxin A" OR "BoNT-B") AND ("efficacy" OR "safety" OR "treatment"). All articles published prior to March 2025 were included. Of 9121 records, 18 studies were included in the final analysis: eight open label studies, five randomized clinical trials, three retrospective studies, and two prospective crossover studies. The studies looked at ET manifesting as upper limb tremor (n = 12), voice tremor (n = 4), and head tremor (n = 2). Among patients receiving BoNT, majority experienced reduction in tremor amplitude and severity and improvement in quality-of-life. Adverse effects were generally dose-dependent and tolerable. Muscle weakness was reported from injections in the upper limb. Hoarseness, breathiness, coughing/choking, dysphagia, and jaw weakness were mostly reported from injections for voice tremors. Lastly, headache, dizziness and dry skin were reported from injections for head tremors. BoNT injections demonstrated effectiveness in reducing tremor severity and is a promising approach for those experiencing symptoms that are refractory to conventional oral medications. Further controlled trials are necessary to confirm these observations.

Background Facial wrinkles, caused by aging and repetitive muscle contractions, are commonly treated with botulinum neurotoxin type A (BoNT-A). However, excessive toxin diffusion can cause side-effects like muscle weakness. Objectives This study aimed to compare the diffusion, efficacy, and safety of letibotulinum toxin A with two other BoNT-A products for treating forehead wrinkles. Methods In a double-blind, randomized, split-face controlled trial, 20 participants with moderate-to-severe horizontal forehead wrinkles received letibotulinum toxin A on one side and prabotulinum or onabotulinum toxin A on the other. The primary outcome was diffusion profile assessed via anhidrosis area (iodine-starch test at 2 weeks); secondary outcomes included wrinkle reduction, assessed by photographic analysis, and safety. Results Results show that letibotulinum toxin A exhibited diffusion patterns and wrinkle-reduction efficacy comparable to the control products. No statistically significant differences were observed be-tween the groups for the primary or secondary outcomes. Conclusion Letibotulinum toxin A is a safe, effective alternative for wrinkle treatment, potentially minimizing excessive diffusion risks and related side effects, making it a valuable addition to available treatment options.

To evaluate the effectiveness of repeated botulinum neurotoxin A (BoNT-A) injections on gross motor function over 3 years in ambulant children with spastic cerebral palsy (CP). A prospective observational cohort study of 124 participants was conducted comparing outcomes in children (aged 2-6 years) with spastic CP functioning in Gross Motor Function Classification System (GMFCS) levels I to III who did and did not receive BoNT-A. The primary outcome was the 66-item Gross Motor Function Measure (GMFM-66), assessed at baseline and annually over 3 years. Secondary outcomes included passive ankle dorsiflexion with knee extended (PADKE) and several measures of activity and participation. A total of 117 participants (94%), consisting of 61 cases and 56 comparisons, were assessed on the GMFM-66 at a follow-up of 1 year or longer, with 106 (85%) assessed at year 3. There were no significant differences in mean GMFM-66 scores adjusted for baseline differences between groups over time (β̂group = 0.92, standard error [SE] = 0.81, 95% confidence interval [CI] = -0.66 to 2.50; p = 0.256). A difference in PADKE favouring the comparison group was observed (β̂group = -4.17, SE = 1.58, 95% CI = -7.27 to -1.08; p = 0.009), approaching the 5° minimally important difference. Repeated BoNT-A injections over 3 years were not associated with improvements in gross motor function or passive ankle dorsiflexion range in young children with spastic CP functioning in GMFCS levels I to III compared to a comparison group not receiving BoNT-A.

Managing Botulinum Toxin A Dosing for Laryngeal Dystonia.

Pain in dystonia is closely related neurological disorders, with significant impact on patients' quality of life. This study aims to conduct a bibliometric analysis to examine the development trends, research hotspots, and future directions in the field of pain in dystonia. Publications from 1981 to 2025 were retrieved from the Web of Science Core Collection database. Bibliometric data were analyzed using the R package "Bibliometrix," VOSviewer, and CiteSpace. A total of 1225 articles were included in this study. The USA had the highest publication volume (329), followed by Germany (103) and Italy (87). The most productive institution was the University of London with 123 publications. Movement Disorders was one of the most influential journals in its field. Key authors in the field included Jankovic Joseph, Marinus Johan, and Van Hilten Jacobus J. The high-frequency keywords were "double-blind," "efficacy," and "spasmodic torticollis." Keywords burst analysis showed emerging interests in "blepharospasm," "botulinum neurotoxin," "quality of life," "diagnosis," "management," and "neurotoxin." This bibliometric study quantitatively analyzed research trends in pain in dystonia, identifying key contributors, hotspots, and emerging trends. Keywords result reflected the growing interest in improving patient outcomes through better diagnostic techniques and therapeutic interventions aimed at alleviating symptoms and enhancing the quality of life for individuals affected by this condition.

Botulinum neurotoxins (BoNT) are the most potent protein toxins for humans, yet how BoNT-Light Chain/A1 (LC/A1) journeys to cleave intracellular SNAP-25 is understudied. Here we use a cell-based assay to measure cytosolic EGFP-LC/A1 intracellular trafficking and SNAP-25 cleavage in Neuro-2A cells. Intracellular LC/A1 associated on microtubules and co-localized with Rab GTPases involved in fast synaptic vesicles and endosome recycling. Multiple Dominant Negative (DN) Rabs GTPases involved in fast synaptic vesicles or endosome recycling inhibited LC/A1 trafficking to the intracellular plasma membrane and SNAP-25 cleavage. A cytosolic LC/A1 variant that bound the plasma membrane from the cytosol was insensitive to DNRab GTPases involved in fast synaptic vesicle recycling. LC/A1 traffics on fast synaptic vesicles to the intracellular plasma membrane to cleave SNAP-25. Our data suggest, like Heavy Chain host cell entry and LC catalysis, LC intracellular trafficking to target host substrates can contribute to bacterial toxin potency.