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Toxins as Tools in Neuroscience 2 Cell and Molecular Neuroscience Module 725 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’ - PowerPoint PPT Presentation
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Toxins as Tools in Neuroscience 2
Cell and Molecular Neuroscience
Module 725
Sean Sweeney
From Lecture 1:
How do we know that the synaptic SNARE proteins arethe 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, 13267-13271Ashton, 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: 1102-8Schoch et al., (2001) Science 294: 1015-6Deitcher et al., (1998) J.Neurosci. 18: 2028-39
Review: Scales et al., (2001) 294: 1015-6
Synaptobrevin is not essentialfor fusion, but is essential for rapid fusion and endocytosis(similar data for Botx and synaptobrevin KOs)
Snake Toxins (usually a cocktail of toxins!)
Alpha- Bungarotoxins
Snake presynaptic PLA2 neurotoxinssimilar to cytosolic phospholipase A2
secreted6x disulphide links (v.stable)Ca2+ dependentConverts 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 paralysisFrom administration to death - lag of 1h
Examination of neurotransmitter release propertiesAt neuromuscular junction:
Open circles: evoked releaseOpen triangles: spontaneous
release
Release is Ca2+ dependent(filled triangles: Ca2+ freemedium)
More stimulation decreases the lag phase
Taipoxin intoxication ofMouse hemidiaphragmNMJ.
Note: depletion of vesicles‘omega’ structures at:Muscle fibreSchwann 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 ofexocytosis or endocytosis?
Do snake PLA2toxins bring abouttheir effects by changing the biophysical properties of thesynaptic vesicle orplasma 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 inthe snake genome (a neuromodulator?).An evolutionary mechanism for the production of manySnake toxins? Fry, B.G. et al., (2003)J. Mol. Evol. 57:110-129
Alpha-bungarotoxin: member of ‘three-finger-toxin’ family
QuickTime™ and aTIFF (Uncompressed) decompressor
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Alpha-bungarotoxin binds to the nicotinic acetylcholine receptor
Red= alpha-bungarotoxin
But does it block it?
QuickTime™ and aTIFF (Uncompressed) decompressor
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Marshall (1981) P.N.A.S78:1948-1952
AChRs are:
Involved in Ach gated fast ionic responses
Pentamers
Each subunit spansthe membrane fourtimes and contributesto the channel pore
A neuronally expressed form of AChR, the alpha-7 receptoris bound and blocked by alpha-bungarotoxin. This form is comprised solely of alpha-7 subunits
Gotti et al.,(1991)P.N.A.S.88:3258-62
AChCa2+
Alpha-bungarotoxin as a neurobiological tool
Alpha-bungarotoxin is a protein, binds tightly to anextracellular target and therefore slow to be cleared and localised in its effects: local injection of alpha-bungarotoxincan be used to ascertain long term effects of receptorblockage.
Plomp, van Kempen and Molenaar (1992) J.Physiol. 458:487-499
Hemidiaphragms injected with alphaBTX every 48h for up to6 weeks and compared to controls:
mEPSPs are recordings of release of one vesicle/quantum.
EPSP is a suprathreshold stimulationOf the nerve inducing the releaseOf multiple vesicles/quanta
Quantal content is EPSP/mEPSP,a measure of the number of vesiclesreleased per stimulus
QuickTime™ and aTIFF (Uncompressed) decompressor
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mEPSPs are a measure ofpostsynaptic function
i.e. a measure of thesize of the postsynaptic receptorfield
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 insize 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 blocksthe movement ofthe action potentialby blocking movementof Na+ into the axon
Binding of TTX tothe Na+ channelblocks the passageof Na+ through the ion channel
The Na+ channel is a Tetramer. Each subunitis a six transmembrane- spanning protein
Tetrodotoxin as a neurobiological tool
Broadie and Bate (1993) activity dependent development of theNeuromuscular synapse during embryogenesis. Neuron 11:607-619
Dispersed GluRs on muscle prior to growth cone arrivalAccumulation of GluRs at site of synaptogenesis on arrival ofGrowth cone/transition to synapse
Is GluR accumulation activity dependent?
Turrigiano et al., (1998) Nature 391: 892-896
Two day treatment of neocortical cellsin culture with TTXor bicuculine (anactivator of firing activity, KCL can beused alternatively)
mEPSPand EPSP sizesare found to beScaled!
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 useof a toxin as a neurobiological molecular scalpel
To Ponder:What are the observable effects of long term blockadeTelling us?