toxins as tools in neuroscience 2 cell and molecular neuroscience module 725 sean sweeney
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Toxins as Tools in Neuroscience 2
Cell and Molecular Neuroscience
Module 725
Sean Sweeney
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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
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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)
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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)
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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
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Taipoxin intoxication ofMouse hemidiaphragmNMJ.
Note: depletion of vesicles‘omega’ structures at:Muscle fibreSchwann cell (?!)
Mechanism of toxicity: external or internal?
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Rigoni et al., (2005) Science 310; 1678-1680
Equivalent effects of snake PLA2 neurotoxins and lysophospholipid/fatty acid mixtures
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Treating synapses with toxin or lysophospholipid/fatty acid Mixtures produce similar effects on synapses.
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Are the ‘omegas’remnants ofexocytosis or endocytosis?
Do snake PLA2toxins bring abouttheir effects by changing the biophysical properties of thesynaptic vesicle orplasma membrane?
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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
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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
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Alpha-bungarotoxin: member of ‘three-finger-toxin’ family
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Alpha-bungarotoxin binds to the nicotinic acetylcholine receptor
Red= alpha-bungarotoxin
But does it block it?
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Marshall (1981) P.N.A.S78:1948-1952
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AChRs are:
Involved in Ach gated fast ionic responses
Pentamers
Each subunit spansthe membrane fourtimes and contributesto the channel pore
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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+
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Alpha-bungarotoxin as a neurobiological tool
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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:
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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
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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
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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?
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Tetrodotoxin
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Tetrodotoxin: a non-peptide toxin
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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
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Tetrodotoxin blocksthe movement ofthe action potentialby blocking movementof Na+ into the axon
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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
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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?
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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!
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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?