Download Ca 2+

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’
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
Schoch et al., (2001) Science 294: 1015-6
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!)
Alpha- 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
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
mEPSP = 0.8mV
mEPSP = 1mV
i.e. a measure of the
size of the
postsynaptic receptor
field
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 movement 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 Na+ channel
is a Tetramer.
Each subunit
is a six
transmembranespanning
protein
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?