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 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: 1102-8Deitcher 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!)

Beta- 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

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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

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

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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 flow ofthe action potentialby blocking movementof Na+ into the axon

Binding of TTX tothe Na+ channelblocks the passageof 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 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?