Toxins as Tools in Neuroscience 2 The Brain in Health and Disease Module 755 Sean Sweeney

From Lecture 1: How do we know that the synaptic SNARE proteins are the only targets of the botulinal toxins? Two competing hypotheses: The ‘metalloproteinase light chain/SNARE substrate’ Hypothesis The Transglutaminase activity hypothesis: Facchiano, F., and Luini, A. (1992) J. Biol. Chem. 267, 13267-13271 Ashton, A. and Dolly, O. (1997) J. Neurochem 68: 649-658

Two hypotheses not necessarily incompatible. What do synaptobrevin knockouts tell us? Deak et al., (2004) Nat. Cell. Biol 6: 1102-8 Deitcher et al., (1998) J.Neurosci. 18: 2028-39 Review: Scales et al., (2001) 294: 1015-6 Synaptobrevin is not essential for fusion, but is essential for rapid fusion and endocytosis (similar data for Botx and synaptobrevin KOs)

Snake Toxins (usually a cocktail of toxins!) Beta- Bungarotoxins Snake presynaptic PLA2 neurotoxins similar to cytosolic phospholipase A2 secreted 6x disulphide links (v.stable) Ca2+ dependent Converts 1,2-diacyl-3-sn phosphoglycerides into fatty acids and lysophospholipids (lysoPL) 1-5 subunits e.g. crotoxin (rattlesnake), ß-bungarotoxin (Krait), taipoxin (taipan)

Intravenous or intraperitoneal injection: Death by respiratory failure caused by paralysis From administration to death - lag of 1h Examination of neurotransmitter release properties At neuromuscular junction: Open circles: evoked release Open triangles: spontaneous release Release is Ca2+ dependent (filled triangles: Ca2+ free medium) More stimulation decreases the lag phase

Taipoxin intoxication of Mouse hemidiaphragm NMJ. Note: depletion of vesicles ‘omega’ structures at: Muscle fibre Schwann cell (?!) Mechanism of toxicity: external or internal?

Rigoni et al., (2005) Science 310; 1678-1680 Equivalent effects of snake PLA2 neurotoxins and lysophospholipid/fatty acid mixtures

Treating synapses with toxin or lysophospholipid/fatty acid Mixtures produce similar effects on synapses.

Are the ‘omegas’ remnants of exocytosis or endocytosis? Do snake PLA2 toxins bring about their effects by changing the biophysical properties of the synaptic vesicle or plasma membrane?

Lethal Doses: Tetanus Toxin: for 70Kg human 175ng Botulinum Toxin: for 70Kg human 90-150ng Tetrodotoxin: 1mg/Kg i.e. 70mg for 70Kg human alpha-Bungarotoxin: 100µg/Kg i.e. 7g for 70Kg human

alpha-bungarotoxin Produced by Taiwanese Many-Banded Krait (Bungarus multicinctus) Only one toxin in a cocktail! (hence low potency?) Chang, CC and Lee CY (1963) Arch. Int. Pharmacodynamics. 144:241-257 May have evolved from another gene present in the snake genome (a neuromodulator?). An evolutionary mechanism for the production of many Snake toxins? Fry, B.G. et al., (2003)J. Mol. Evol. 57:110-129

Alpha-bungarotoxin: member of ‘three-finger-toxin’ family

Alpha-bungarotoxin binds to the nicotinic acetylcholine receptor Red= alpha-bungarotoxin But does it block it?

Marshall (1981) P.N.A.S 78:1948-1952

AChRs are: Involved in Ach gated fast ionic responses Pentamers Each subunit spans the membrane four times and contributes to the channel pore

A neuronally expressed form of AChR, the alpha-7 receptor is bound and blocked by alpha-bungarotoxin. This form is comprised solely of alpha-7 subunits Ca2+ ACh Gotti et al.,(1991)P.N.A.S.88:3258-62

Alpha-bungarotoxin as a neurobiological tool

Alpha-bungarotoxin is a protein, binds tightly to an extracellular target and therefore slow to be cleared and localised in its effects: local injection of alpha-bungarotoxin can be used to ascertain long term effects of receptor blockage. Plomp, van Kempen and Molenaar (1992) J.Physiol. 458:487-499 Hemidiaphragms injected with alphaBTX every 48h for up to 6 weeks and compared to controls:

mEPSPs are recordings of release of one vesicle/quantum. EPSP is a suprathreshold stimulation Of the nerve inducing the release Of multiple vesicles/quanta Quantal content is EPSP/mEPSP, a measure of the number of vesicles released per stimulus

mEPSPs are a measure of postsynaptic function i.e. a measure of the size of the postsynaptic receptor field mEPSP = 0.8mV mEPSP = 1mV

Plomp et al.,: After six weeks alphaBTX treatment: mEPPs were reduced in size by 57% of untreated control Quantal content was increased to 154%!! After a single injection of alphaBTX mEPPs were reduced in size by 60% but no increase in quantal content was observed! At timepoints between acute treatment and 6 weeks with alphaBTX quantal content increased, reaching a plateau Between 20 and 30 days. A mechanism of modulation?

Tetrodotoxin

Tetrodotoxin: a non-peptide toxin

Cultured pufferfish do not produce toxin: acquisition from diet Blue-ringed octopus possess toxin producing bacteria in a specialised salivary gland Member of a group of toxins called Saxitoxins Most poisonings occur from ingestion of poorly prepared Fugu rubripes pufferfish as sushi. Ca. 1 death per year

Tetrodotoxin blocks the flow of the action potential by blocking movement of Na+ into the axon

Binding of TTX to the Na+ channel blocks the passage of Na+ through the ion channel The alpha-subunit of the Na+ channel is a Tetramer. Each homologous subunit is a six trans-membrane- spanning protein

Mutations in the selectivity pore (P-loop) generate resistance to TTX or STX: Selective and adaptive variations

Tetrodotoxin as a neurobiological tool Broadie and Bate (1993) activity dependent development of the Neuromuscular synapse during embryogenesis. Neuron 11: 607-619 Dispersed GluRs on muscle prior to growth cone arrival Accumulation of GluRs at site of synaptogenesis on arrival of Growth cone/transition to synapse Is GluR accumulation activity dependent?

Two day treatment of neocortical cells in culture with TTX or bicuculine (an activator of firing activity, KCL can be used alternatively) mEPSP and EPSP sizes are found to be Scaled! Turrigiano et al., (1998) Nature 391: 892-896

Toxins can be exquisitely precise in their targets (both cellular and intracellular) Toxins can be enzymatic or antagonist/poison Knowing the precise method of action allows the use of a toxin as a neurobiological molecular scalpel To Ponder: What are the observable effects of long term blockade Telling us?