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Toxins as Tools in Neuroscience Cell and Molecular Neuroscience Module 725 Sean Sweeney

Toxins as Tools in Neuroscience Cell and Molecular Neuroscience Module 725 Sean Sweeney. Why study toxins? “Poisons are chemical scalpels for the dissection Of physiological processes” Claude Bernard 1813-1878 Toxins have evolved to incapacitate organisms by attacking

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Toxins as Tools in Neuroscience Cell and Molecular Neuroscience Module 725 Sean Sweeney

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  1. Toxins as Tools in Neuroscience Cell and Molecular Neuroscience Module 725 Sean Sweeney

  2. Why study toxins? “Poisons are chemical scalpels for the dissection Of physiological processes” Claude Bernard 1813-1878 Toxins have evolved to incapacitate organisms by attacking the processes that are most vital to the functions of the organism that is the target of the toxin. By identifying the cellular target of a toxin, we identify a key process in the function of the cell/organism.

  3. Toxins can be produced by pathogens/predatory organisms as a tool in their attack on the host/prey organism (bacterial toxins, snake venom, scorpion venom, spider venom, marine snail venom) Toxins can be produced as a means of defence (poison arrow frog toxins, puffer fish toxin, scorpion fish spine toxin, blue ringed octopus)

  4. Many toxins exert their effects on the nervous system Why?

  5. Case Studies The Clostridial Toxins (Lecture 1) Tetrodotoxin (Lecture 2) Bungarotoxin (Lecture2)

  6. Clostridia interactions with humans C. botulinum : botulism, food poisoning C. difficile : pseudomembranous colitis (fluid accumulation in bowel) C. perfringens: gas gangrene, uterine infections C. tetanus : Paralysis (lockjaw) from infected wounds All are Gram positive spore forming bacteria, often found in soil, preferring anaerobic conditions for growth. All produce toxins.

  7. C.tetanus and C.botulinum: paralysis but by different means Botulinum intoxication Avian botulism ‘Floppy baby syndrome’ Flaccid paralysis

  8. “When tetanus occurs, the jaws become as hard as wood, And patients cannot open their mouths. Their eyes shed Tears and look awry, their backs become rigid, and they Cannot adduct their legs; similarly, not their arms either. The patient’s face becomes red, he suffers great pain and, When he is on the point of death, he vomits drink gruel And phlegm through his nostrils. The patient generally Dies on the third, fifth, seventh or fourteenth day; if he Survives for that many, he recovers.” Hippocrates, Diseases III (4th Century B.C.)

  9. Tetanus intoxication induces a rigid paralysis ‘opistotonus’ ‘risus sardonicus’

  10. C. tetani and C. botulinum produce toxins that can account for all of the observed effects of infection by these organisms C. tetani Tetanus toxin (TeTx) C. Botulinum strain A Botulinum toxin A (BoTxA) strain B Botulinum toxin B (BoTxB) strain C Botulinum toxin C (BoTxC) strain D Botulinum toxin D (BoTxD) strain E Botulinum toxin E (BoTxE) strain F Botulinum toxin F (BoTxF) strain G Botulinum toxin G (BoTxG) A,B,E and F affect humans C and D affect birds and some mammals G has never been identified as causing disease. Isolated from soil in Argentina

  11. Clostridial neurotoxins have domain structure homology All are secreted as 150kD Single chain proteins with a single di-sulfide bond. Toxin is cleaved into two chains, heavy chain (H-chain) and light chain (L-chain).

  12. The toxins have a cellular and intracellular site of action. Toxins must therefore: 1.Bind to their target cell 2.Translocate to the interior of the cell 3.Find and modify their intracellular target

  13. Toxin consists of three main functional domains: HC : Extracellular binding domain HN : Translocation domain LC : Enzymatic domain

  14. The Clostridial toxins bind to their target cells via the HC Domain. The HC domain binds to ganglioside targets Gangliosides are carbohydrate modified sphingolipids found on the external leaflet of the plasma membrane

  15. Clostridial toxins intracellularise via endosytosis and a pH dependent membrane translocation

  16. Mochida et al.,(1990) P.N.A.S. Injection of light chain mRNA of Clostridial toxins inhibits synaptic transmission. Clostridial toxin induces Accumulation of synaptic vesicles at active zones

  17. The synaptic vesicle cycle

  18. Clostridial toxins block synaptic transmission. Why do they induce such different paralytic outcomes? The site of action for BoTx lies in the motorneurons

  19. The site of action for TeTx lies in the inhibitory spinal interneurons

  20. BoTx binds to and is internalised by motorneurons via endocytosis. The toxin exits from the endosome and blocks synaptic transmission TeTx binds to motorneurons, is retrogradely transported along the axon within a endosomal vesicle. TeTx is then trans-synaptically transported into an inhibitory Interneuron, released into the cytoplasm and blocks synaptic transmission.

  21. The Clostridial toxins share metalloproteinase sequence homology within the light chain. This suggested that Clostridial toxins might cleave their Intracellular target

  22. How do Clostridial toxins block the synaptic vesicle cycle?

  23. Cleavage of the synaptic vesicle protein synaptobrevin/ VAMP correlates with tetanus intoxication and loss of synaptic transmission

  24. Clostridial toxins cleave Proteins associated with the Synaptic vesicle and the Plasma membrane: VAMP/synaptobrevin (on the SV) Syntaxin and SNAP-25 on the PM

  25. Cleavage is extremely specific. BUT, toxins can cross inhibit! Indicates similar structural requirements for toxin binding

  26. Syntaxin TM SNARE BoTx Synaptobrevin/VAMP SNARE SNARE TM BoTx BoTx Toxin cleavage sites are preceded by structural motifs necessary for recognition of target by toxin. SNARE motifs are the basis of cross-inhibition and give the name To the protein classification: SNARE proteins (v,t,Q and R)

  27. The three SNARE Proteins form an SDS resistant complex that is resistant to toxin cleavage.

  28. …and bind two proteins previously identified as essential to vesicle traffic throughout the cell, NSF and SNAP NSF is an ATPase

  29. The SNARE proteins plus NSF and SNAP form a 20S Complex. Importance of SNARE protein integrity for exocytosis in conjunction with the energy input requirement indicates a key point of regulation for vesicle fusion

  30. SNARE proteins have orthologs in yeast that regulate Vesicle trafficking fusion steps suggesting the mechanism of vesicle fusion is ancient (preceeding the evolution of neurotransmission) Shekman and Novick (2004) Cell 116(suppl)S13-15 Novick, Field and Sheckman (1980) Cell 21; 105-215

  31. Other SNARE-like proteins regulate vesicle traffic in other vesicle fusion steps throughout the cell! Are SNAREs a general mechanism? The SNARE hypothesis Rothman and Warren (1992) Curr. Biol. 116:135-146

  32. The study of Clostridial toxin action has revealed conserved Mechanisms that regulate vesicle fusion and traffic

  33. Clostridial Toxins as tools in cell biology: Testing of roles of individual SNARE proteins in secretory processes. Clostridial Toxins as therapeutic tools: Blepharospasm Facial hemispasm Excessive palm and foot perspiration Botox treatment Clostridial toxins as agents of warfare.

  34. Relax, its only Botox…….

  35. Clostridial Toxins as Tools for Behavioural Studies Or: I’d rather have a bottle in front of me to a frontal lobotomy Mapping Neural circuits in flies: What circuits underlie particular behaviours? How do we Identify them?

  36. The GAL4/UAS system in flies is a binary system that allows generation and expression of toxic transgenes in any tissue of choice UAS-eyeless/dpp-GAL4 We can use this system to express TeTxLC in flies as a tool to study behaviour (Sweeney et al (1995) Neuron 14, 341-351 Martin, Keller and Sweeney (2002) Advances in Genetics 47, 1-47)

  37. There are two synaptobrevins In flies. Only one is cleaved by TeTxLC (Sweeney et al 1995)

  38. Muscle-TeTxLC Neuronal-TeTxLC Synaptotagmin staining n-synaptobrevin staining Neuronal expression of TeTxLC abolishes n-synaptobrevin staining (Sweeney et al., ‘95)

  39. Neuronal expression of TeTxLC abolishes neuromuscular synaptic transmission (Sweeney et al, ‘95)

  40. Can we use UAS-TeTxLC as a tool to study behaviour?

  41. Tracey et al (2003) Painless, a Drosophila gene essential for nociception. Cell, 113, 261-273

  42. Painless protein is expressed in the PNS. Expression of TeTxLC in the painless expressing cells phenocopies the painless mutant

  43. GAL4/UAS-TeTxLC can be used to effectively map and define neural circuits underlying specific behaviours. Next week: Dodgy sushi and neurotoxicity: tetrodotoxin Banded kraits and mammalian NMJs: alpha-bungarotoxin

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