Botulinum neurotoxins (BoNTs) and tetanus toxin (TeTX) are the deadliest biological substances that cause botulism and tetanus, respectively. Their astonishing potency and capacity to enter neurons and interfere with neurotransmitter release at presynaptic terminals have attracted much interest in experimental neurobiology and clinical research. Fused with reporter proteins or labelled with fluorophores, BoNTs and TeTX and their non-toxic fragments also offer remarkable opportunities to visualize cellular processes and functions in neurons and synaptic connections. This study presents the state-of-the-art optical probes derived from BoNTs and TeTX and discusses their applications in molecular and synaptic biology and neurodevelopmental research. It reviews the principles of the design and production of probes, revisits their applications with advantages and limitations and considers prospects for future improvements. The versatile characteristics of discussed probes and reporters make them an integral part of the expanding toolkit for molecular neuroimaging, promoting the discovery process in neurobiology and translational neurosciences.

Chair of Biological Imaging at the Central Institute for Translational Cancer Research School of Medicine Technical University of Munich 81675 Munich Germany

Department of Medical Genetics 3rd Faculty of Medicine Charles University Ruská 87 10000 Prague Czech Republic

DZHK Partner Site Munich Heart Alliance Munich Germany

Faculty of Engineering and Science University of Greenwich London Chatham Maritime Kent ME4 4TB UK

Faculty of Medicine Ivane Javakhishvili Tbilisi State University 0159 Tbilisi Georgia

Institute of Biological and Medical Imaging and Healthcare Helmholtz Zentrum München 85764 Neuherberg Germany

Munich Institute of Robotics and Machine Intelligence Technical University of Munich 80992 Munich Germany

Zobrazit více v PubMed

Ntziachristos V (2010) Going deeper than microscopy: the optical imaging frontier in biology. Nat Methods 7:603–614 PubMed

Yoon S, Cheon SY, Park S et al (2022) Recent advances in optical imaging through deep tissue: imaging probes and techniques. Biomater Res 26:57 PubMed PMC

Fernandez-Suarez M, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9:929–943 PubMed

Linghu C, Johnson SL, Valdes PA et al (2020) Spatial multiplexing of fluorescent reporters for imaging signaling network dynamics. Cell 183(1682–1698):e1624 PubMed PMC

Shemesh OA, Linghu C, Piatkevich KD et al (2020) Precision calcium imaging of dense neural populations via a cell-body-targeted calcium indicator. Neuron 107(470–486):e411 PubMed PMC

Westphal V, Rizzoli SO, Lauterbach MA, Kamin D, Jahn R, Hell SW (2008) Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320:246–249 PubMed

Yu XF, Sun Z, Li M et al (2010) Neurotoxin-conjugated upconversion nanoprobes for direct visualization of tumors under near-infrared irradiation. Biomaterials 31:8724–8731 PubMed

Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544 PubMed

Baetke SC, Lammers T, Kiessling F (2015) Applications of nanoparticles for diagnosis and therapy of cancer. Brit J Radiol 88(1054):20150207 PubMed PMC

Ovsepian SV, O’Leary VB, Ntziachristos V, Dolly JO (2016) Circumventing brain barriers: nanovehicles for retroaxonal therapeutic delivery. Trends Mol Med 22:983–993 PubMed

Thangam R, Paulmurugan R, Kang H (2022) Functionalized nanomaterials as tailored theranostic agents in brain imaging. Nanomaterials 12(1):18 PubMed PMC

Thiruppathi R, Mishra S, Ganapathy M, Padmanabhan P, Gulyas B (2017) Nanoparticle functionalization and its potentials for molecular imaging. Adv Sci (Weinh) 4:1600279 PubMed PMC

Ergen PH, Shorter S, Ntziachristos V, Ovsepian SV (2023) Neurotoxin-derived optical probes for biological and medical imaging. Mol Imaging Biol 25:799–814 PubMed PMC

Jenner R, Undheim E (2017) Venom: the secrets of nature’s deadliest weapon. Smithsonian

Jurenas D, Fraikin N, Goormaghtigh F, Van Melderen L (2022) Biology and evolution of bacterial toxin-antitoxin systems. Nat Rev Microbiol 20:335–350 PubMed

van Thiel J, Khan MA, Wouters RM et al (2022) Convergent evolution of toxin resistance in animals. Biol Rev Camb Philos Soc 97:1823–1843 PubMed PMC

Wilcox C (2017) Venomous: How Earth’s Deadliest Creatures Mastered Biochemistry. Farrar, Straus and Giroux

Pirazzini M, Montecucco C, Rossetto O (2022) Toxicology and pharmacology of botulinum and tetanus neurotoxins: an update. Arch Toxicol 96:1521–1539 PubMed PMC

Dong M, Masuyer G, Stenmark P (2019) Botulinum and Tetanus Neurotoxins. Annu Rev Biochem 88:811–837 PubMed PMC

Rossetto O, Montecucco C (2019) Tables of toxicity of botulinum and tetanus neurotoxins. Toxins (Basel) 11(12):686 PubMed PMC

Montecucco C, Schiavo G (1995) Structure and function of tetanus and botulinum neurotoxins. Q Rev Biophys 28:423–472 PubMed

Megighian A, Pirazzini M, Fabris F, Rossetto O, Montecucco C (2021) Tetanus and tetanus neurotoxin: From peripheral uptake to central nervous tissue targets. J Neurochem 158:1244–1253 PubMed

Binz T, Rummel A (2009) Cell entry strategy of clostridial neurotoxins. J Neurochem 109:1584–1595 PubMed

Montal M (2010) Botulinum neurotoxin: a marvel of protein design. Annu Rev Biochem 79:591–617 PubMed

Schiavo G, Benfenati F, Poulain B et al (1992) Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359:832–835 PubMed

Ovsepian SV, O’Leary VB, Ayvazyan NM, Al-Sabi A, Ntziachristos V, Dolly JO (2019) Neurobiology and therapeutic applications of neurotoxins targeting transmitter release. Pharmacol Ther 193:135–155 PubMed

Dolly JO, Meng J, Wang J et al (2009) Multiple Steps in the Blockade of Exocytosis by Botulinum Neurotoxins. In: Toxin B (ed) Jankovic J, Albanese A, Atassi MZ, Dolly JO, Hallett M, Mayer NH. Saunders, W.B, pp 1-14.e11

Machamer JB, Vazquez-Cintron EJ, Stenslik MJ et al (2023) Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity. Front Cell Neurosci 17:1226194 PubMed PMC

Meunier FA, Schiavo G, Molgo J (2002) Botulinum neurotoxins: from paralysis to recovery of functional neuromuscular transmission. J Physiol Paris 96:105–113 PubMed

Johnson EA, Montecucco C (2008) Chapter 11 Botulism. Elsevier

Dolly JO, Black J, Williams RS, Melling J (1984) Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization. Nature 307:457–460 PubMed

Fishman PS (2009) Tetanus Toxin. In: Toxin B (ed) Jankovic J, Albanese A, Atassi MZ, Dolly JO, Hallett M, Mayer NH. Saunders, W.B, pp 406-424.e401

Ovsepian SV, Bodeker M, O’Leary VB, Lawrence GW, Oliver Dolly J (2015) Internalization and retrograde axonal trafficking of tetanus toxin in motor neurons and trans-synaptic propagation at central synapses exceed those of its C-terminal-binding fragments. Brain Struct Funct 220:1825–1838 PubMed

Dolly JO, Lawrence GW, Meng J, Wang J, Ovsepian SV (2009) Neuro-exocytosis: botulinum toxins as inhibitory probes and versatile therapeutics. Curr Opin Pharmacol 9:326–335 PubMed

Jankovic J (2004) Botulinum toxin in clinical practice. J Neurol Neurosurg Psychiatry 75:951–957 PubMed PMC

Jankovic J, Albanese A, Atassi MZ, Dolly JO, Hallett M, Mayer NH (2009) Botulinum toxin: therapeutic clinical practice and science. Elsevier Health Sciences

Meng J, Ovsepian SV, Wang J et al (2009) Activation of TRPV1 mediates calcitonin gene-related peptide release, which excites trigeminal sensory neurons and is attenuated by a retargeted botulinum toxin with anti-nociceptive potential. J Neurosci 29:4981–4992 PubMed PMC

Dressler D, Johnson EA (2022) Botulinum toxin therapy: past, present and future developments. J Neural Transm (Vienna) 129:829–833 PubMed PMC

Keller JE, Neale EA, Oyler G, Adler M (1999) Persistence of botulinum neurotoxin action in cultured spinal cord cells. FEBS Lett 456:137–142 PubMed

Keller JE, Neale EA (2001) The role of the synaptic protein snap-25 in the potency of botulinum neurotoxin type A. J Biol Chem 276:13476–13482 PubMed

Fernandez-Salas E, Ho H, Garay P, Steward LE, Aoki KR (2004) Is the light chain subcellular localization an important factor in botulinum toxin duration of action? Mov Disord 19(Suppl 8):S23-34 PubMed

Fernandez-Salas E, Steward LE, Ho H et al (2004) Plasma membrane localization signals in the light chain of botulinum neurotoxin. Proc Natl Acad Sci USA 101:3208–3213 PubMed PMC

Chen S, Barbieri JT (2011) Association of botulinum neurotoxin serotype A light chain with plasma membrane-bound SNAP-25. J Biol Chem 286:15067–15072 PubMed PMC

Cai BB, Francis J, Brin MF, Broide RS (2017) Botulinum neurotoxin type A-cleaved SNAP25 is confined to primary motor neurons and localized on the plasma membrane following intramuscular toxin injection. Neuroscience 352:155–169 PubMed

Pellett S, Tepp WH, Whitemarsh RC, Bradshaw M, Johnson EA (2015) In vivo onset and duration of action varies for botulinum neurotoxin A subtypes 1–5. Toxicon 107:37–42 PubMed PMC

Pellett S, Bradshaw M, Tepp WH et al (2018) The light chain defines the duration of action of botulinum toxin serotype A subtypes. mBio 9(2):10–1128 PubMed PMC

Fornasiero EF, Mandad S, Wildhagen H et al (2018) Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions. Nat Commun 9:4230 PubMed PMC

Mathieson T, Franken H, Kosinski J et al (2018) Systematic analysis of protein turnover in primary cells. Nat Commun 9:689 PubMed PMC

Wang J, Zurawski TH, Meng J et al (2011) A dileucine in the protease of botulinum toxin A underlies its long-lived neuroparalysis: transfer of longevity to a novel potential therapeutic. J Biol Chem 286:6375–6385 PubMed PMC

Pirazzini M, Rossetto O, Eleopra R, Montecucco C (2017) Botulinum neurotoxins: biology, pharmacology, and toxicology. Pharmacol Rev 69:200–235 PubMed PMC

Tsai YC, Maditz R, Kuo CL et al (2010) Targeting botulinum neurotoxin persistence by the ubiquitin-proteasome system. Proc Natl Acad Sci USA 107:16554–16559 PubMed PMC

Xie S, Xu H, Shan XF, Cai ZG (2019) Botulinum toxin type A interrupts autophagic flux of submandibular gland. Biosci Rep 39(7):BSR20190035 PubMed PMC

Vagin O, Tokhtaeva E, Garay PE et al (2014) Recruitment of septin cytoskeletal proteins by botulinum toxin A protease determines its remarkable stability. J Cell Sci 127:3294–3308 PubMed PMC

Wang T, Martin S, Papadopulos A et al (2015) Control of autophagosome axonal retrograde flux by presynaptic activity unveiled using botulinum neurotoxin type a. J Neurosci 35:6179–6194 PubMed PMC

Goodnough MC, Oyler G, Fishman PS et al (2002) Development of a delivery vehicle for intracellular transport of botulinum neurotoxin antagonists. FEBS Lett 513:163–168 PubMed

Harper CB, Martin S, Nguyen TH et al (2011) Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J Biol Chem 286:35966–35976 PubMed PMC

Ho M, Chang LH, Pires-Alves M et al (2011) Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting. Protein Eng Des Sel 24:247–253 PubMed PMC

Colasante C, Rossetto O, Morbiato L, Pirazzini M, Molgo J, Montecucco C (2013) Botulinum neurotoxin type A is internalized and translocated from small synaptic vesicles at the neuromuscular junction. Mol Neurobiol 48:120–127 PubMed

Benoit RM, Frey D, Hilbert M et al (2014) Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A. Nature 505:108–111 PubMed

Dong M, Yeh F, Tepp WH et al (2006) SV2 is the protein receptor for botulinum neurotoxin A. Science 312:592–596 PubMed

Joensuu M, Syed P, Saber SH et al (2023) Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG-Syt1-SV2 plasma membrane nanocluster for synaptic vesicle entry. EMBO J 42:e112095 PubMed PMC

Takamori S, Holt M, Stenius K et al (2006) Molecular anatomy of a trafficking organelle. Cell 127:831–846 PubMed

Verderio C, Grumelli C, Raiteri L et al (2007) Traffic of botulinum toxins A and E in excitatory and inhibitory neurons. Traffic 8:142–153 PubMed

Bercsenyi K, Giribaldi F, Schiavo G (2013) The elusive compass of clostridial neurotoxins: deciding when and where to go? Curr Top Microbiol Immunol 364:91–113 PubMed

Antonucci F, Rossi C, Gianfranceschi L, Rossetto O, Caleo M (2008) Long-distance retrograde effects of botulinum neurotoxin A. J Neurosci 28:3689–3696 PubMed PMC

Lawrence GW, Ovsepian SV, Wang J, Aoki KR, Dolly JO (2012) Extravesicular intraneuronal migration of internalized botulinum neurotoxins without detectable inhibition of distal neurotransmission. Biochem J 441:443–452 PubMed

Bohnert S, Schiavo G (2005) Tetanus toxin is transported in a novel neuronal compartment characterized by a specialized pH regulation. J Biol Chem 280:42336–42344 PubMed

Lalli G, Gschmeissner S, Schiavo G (2003) Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons. J Cell Sci 116:4639–4650 PubMed

Roux S, Colasante C, Saint Cloment C et al (2005) Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions. Mol Cell Neurosci 30:572–582 PubMed

Roesl C, Evans ER, Dissanayake KN et al (2021) Confocal endomicroscopy of neuromuscular junctions stained with physiologically inert protein fragments of tetanus toxin. Biomolecules 11(10):1499 PubMed PMC

Matsuda K, Yoshida M, Kawakami K, Hibi M, Shimizu T (2017) Granule cells control recovery from classical conditioned fear responses in the zebrafish cerebellum. Sci Rep 7:11865 PubMed PMC

Kissa K, Mordelet E, Soudais C et al (2002) In vivo neuronal tracing with GFP-TTC gene delivery. Mol Cell Neurosci 20:627–637 PubMed

Sakurai T, Nagata R, Yamanaka A et al (2005) Input of orexin/hypocretin neurons revealed by a genetically encoded tracer in mice. Neuron 46:297–308 PubMed

Maskos U, Kissa K, St Cloment C, Brulet P (2002) Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice. Proc Natl Acad Sci USA 99:10120–10125 PubMed PMC

Harms KJ, Tovar KR, Craig AM (2005) Synapse-specific regulation of AMPA receptor subunit composition by activity. J Neurosci 25:6379–6388 PubMed PMC