Case Studies The Clostridial Toxins

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Toxins as Tools in Neuroscience
Cell and Molecular Neuroscience
Module 725
Sean Sweeney
Why study toxins?
“Poisons are chemical scalpels for the dissection
Of physiological processes”
Claude Bernard 1813-1878
Toxins have evolved to incapacitate organisms by attacking
the processes that are most vital to the functions of the
organism that is the target of the toxin.
By identifying the cellular target of a toxin, we identify a key
process in the function of the cell/organism.
Toxins can be produced by pathogens/predatory organisms
as a tool in their attack on the host/prey organism
(bacterial toxins, snake venom, scorpion venom,
spider venom, marine snail venom)
Toxins can be produced as a means of defence
(poison arrow frog toxins, puffer fish toxin,
scorpion fish spine toxin, blue ringed octopus)
Many toxins exert their effects on the nervous system
Why?
Case Studies
The Clostridial Toxins (Lecture 1)
Tetrodotoxin (Lecture 2)
Bungarotoxin (Lecture2)
Clostridia interactions with humans
C. botulinum
C. difficile
:
:
botulism, food poisoning
pseudomembranous colitis
(fluid accumulation in bowel)
C. perfringens:
gas gangrene, uterine infections
C. tetanus
Paralysis (lockjaw) from infected
wounds
:
All are Gram positive spore forming bacteria,
often found in soil, preferring anaerobic conditions for
growth. All produce toxins.
C.tetanus and C.botulinum: paralysis but by different means
Botulinum intoxication
‘Floppy baby syndrome’
Avian botulism
Flaccid paralysis
“When tetanus occurs, the jaws become as hard as wood,
And patients cannot open their mouths. Their eyes shed
Tears and look awry, their backs become rigid, and they
Cannot adduct their legs; similarly, not their arms either.
The patient’s face becomes red, he suffers great pain and,
When he is on the point of death, he vomits drink gruel
And phlegm through his nostrils. The patient generally
Dies on the third, fifth, seventh or fourteenth day; if he
Survives for that many, he recovers.”
Hippocrates, Diseases III (4th Century B.C.)
Tetanus intoxication induces a rigid paralysis
‘opistotonus’
‘risus sardonicus’
C. tetani and C. botulinum produce toxins that can account
for all of the observed effects of infection by these organisms
C. tetani
C. Botulinum
Tetanus toxin (TeTx)
strain A
strain B
strain C
strain D
strain E
strain F
strain G
Botulinum toxin A (BoTxA)
Botulinum toxin B (BoTxB)
Botulinum toxin C (BoTxC)
Botulinum toxin D (BoTxD)
Botulinum toxin E (BoTxE)
Botulinum toxin F (BoTxF)
Botulinum toxin G (BoTxG)
A,B,E and F affect humans
C and D affect birds and some mammals
G has never been identified as causing disease. Isolated
from soil in Argentina
Clostridial neurotoxins
have domain structure
homology
All are secreted as 150kD
Single chain proteins
with a single di-sulfide
bond.
Toxin is cleaved into two
chains, heavy chain (Hchain) and light chain (Lchain).
The toxins have a cellular and intracellular site of action.
Toxins must therefore:
1.Bind to their target cell
2.Translocate to the interior of the cell
3.Find and modify their intracellular target
Toxin consists of three main functional domains:
HC
HN
LC
: Extracellular binding domain
: Translocation domain
: Enzymatic domain
The Clostridial toxins bind to their target cells via the HC
Domain. The HC domain binds to ganglioside targets
Gangliosides are carbohydrate modified sphingolipids found
on the external leaflet of the plasma membrane
Clostridial toxins intracellularise via endosytosis and a
pH dependent membrane translocation
Mochida et al.,(1990) P.N.A.S.
Injection of light chain mRNA of
Clostridial toxins inhibits
synaptic transmission.
Clostridial toxin induces
Accumulation of synaptic vesicles
at active zones
The synaptic vesicle cycle
Clostridial toxins block synaptic transmission.
Why do they induce such different paralytic outcomes?
The site of action for BoTx lies in the motorneurons
The site of action for TeTx lies in the inhibitory spinal
interneurons
BoTx binds to and is internalised
by motorneurons via endocytosis.
The toxin exits from the endosome
and blocks synaptic transmission
TeTx binds to motorneurons, is
retrogradely transported along the
axon within a endosomal vesicle.
TeTx is then trans-synaptically
transported into an inhibitory
Interneuron, released into the
cytoplasm and blocks
synaptic transmission.
The Clostridial toxins share metalloproteinase sequence
homology within the light chain.
This suggested that Clostridial toxins might cleave their
Intracellular target
How do Clostridial toxins block the synaptic vesicle cycle?
Cleavage of the synaptic
vesicle protein synaptobrevin/
VAMP correlates with tetanus
intoxication and loss of
synaptic transmission
Clostridial toxins cleave
Proteins associated with the
Synaptic vesicle and the
Plasma membrane:
VAMP/synaptobrevin (on the
SV)
Syntaxin and SNAP-25 on
the PM
Cleavage is extremely specific.
BUT, toxins can cross inhibit!
Indicates similar structural requirements for
toxin binding
Syntaxin
TM
SNARE
BoTx
Synaptobrevin/VAMP
SNARE
TM
BoTx
SNARE
BoTx
Toxin cleavage sites are preceded by structural motifs
necessary for recognition of target by toxin. SNARE
motifs are the basis of cross-inhibition and give the name
To the protein classification: SNARE proteins (v,t,Q and R)
The three SNARE Proteins form an SDS resistant complex
that is resistant to toxin cleavage.
…and bind two proteins previously identified as essential
to vesicle traffic throughout the cell, NSF and SNAP
NSF is an ATPase
The SNARE proteins plus NSF and SNAP form a 20S
Complex.
Importance of SNARE protein integrity for exocytosis
in conjunction with the energy input requirement indicates
a key point of regulation for vesicle fusion
SNARE proteins have orthologs in yeast that regulate
Vesicle trafficking fusion steps suggesting
the mechanism of vesicle fusion is ancient
(preceeding the evolution of neurotransmission)
Shekman and Novick (2004) Cell 116(suppl)S13-15
Novick, Field and Sheckman (1980) Cell 21; 105-215
Other SNARE-like proteins regulate vesicle traffic in other
vesicle fusion steps throughout the cell! Are SNAREs a
general mechanism?
The SNARE hypothesis
Rothman and Warren (1992) Curr. Biol. 116:135-146
The study of Clostridial toxin action has revealed conserved
Mechanisms that regulate vesicle fusion and traffic
Clostridial Toxins as tools in cell biology:
Testing of roles of individual SNARE proteins in secretory
processes.
Clostridial Toxins as therapeutic tools:
Blepharospasm
Facial hemispasm
Excessive palm and foot perspiration
Botox treatment
Clostridial toxins as agents of warfare.
Relax, its only Botox…….
Clostridial Toxins as Tools for Behavioural Studies
Or:
I’d rather have a bottle in front of me to a frontal lobotomy
Mapping Neural circuits in flies:
What circuits underlie particular behaviours? How do we
Identify them?
The GAL4/UAS system in flies is a binary system that
allows generation and expression of toxic transgenes
in any tissue of choice
UAS-eyeless/dpp-GAL4
We can use this system to express TeTxLC in flies as
a tool to study behaviour
(Sweeney et al (1995) Neuron 14, 341-351
Martin, Keller and Sweeney (2002) Advances in Genetics
47, 1-47)
There are two
synaptobrevins
In flies. Only one
is cleaved by
TeTxLC
(Sweeney et al 1995)
Muscle-TeTxLC
Neuronal-TeTxLC
Synaptotagmin
staining
n-synaptobrevin
staining
Neuronal expression of TeTxLC
abolishes n-synaptobrevin staining
(Sweeney et al., ‘95)
Neuronal expression of
TeTxLC abolishes
neuromuscular synaptic
transmission
(Sweeney et al, ‘95)
Can we use UAS-TeTxLC as a tool to study behaviour?
Tracey et al (2003) Painless,
a Drosophila gene essential
for nociception.
Cell, 113, 261-273
Painless protein is expressed in the PNS.
Expression of TeTxLC in the painless
expressing cells phenocopies the painless
mutant
GAL4/UAS-TeTxLC can be used to effectively map
and define neural circuits underlying specific
behaviours.
Next week:
Dodgy sushi and neurotoxicity: tetrodotoxin
Banded kraits and mammalian NMJs: alpha-bungarotoxin
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