Toxins as Tools in Neuroscience

Cell and Molecular Neuroscience

Module 725

Sean Sweeney

Why study toxins?

“Poisons are chemical scalpels for the dissectionOf physiological processes”

Claude Bernard 1813-1878

Toxins have evolved to incapacitate organisms by attackingthe 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 organismsas 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 : botulism, food poisoning

C. difficile : pseudomembranous colitis(fluid accumulation in bowel)

C. perfringens: gas gangrene, uterine infections

C. tetanus : Paralysis (lockjaw) from infectedwounds

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

Flaccid paralysis

‘Floppy baby syndrome’ Avian botulism

“When tetanus occurs, the jaws become as hard as wood,And patients cannot open their mouths. Their eyes shedTears 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 generallyDies on the third, fifth, seventh or fourteenth day; if heSurvives for that many, he recovers.”

Hippocrates, Diseases III (4th Century B.C.)

Tetanus intoxication induces a rigid paralysis

‘risus sardonicus’

‘opistotonus’

C. tetani and C. botulinum produce toxins that can accountfor all of the observed effects of infection by these organisms

C. tetani Tetanus toxin (TeTx)

C. Botulinum strain A Botulinum toxin A (BoTxA)strain B Botulinum toxin B (BoTxB)strain C Botulinum toxin C (BoTxC)strain D Botulinum toxin D (BoTxD)strain E Botulinum toxin E (BoTxE)strain F Botulinum toxin F (BoTxF)strain G Botulinum toxin G (BoTxG)

A,B,E and F affect humansC and D affect birds and some mammalsG has never been identified as causing disease. Isolated from soil in Argentina

Clostridial neurotoxinshave domain structure homology

All are secreted as 150kDSingle chain proteins with a single di-sulfide bond. Toxin is cleaved into two chains, heavy chain (H-chain) and light chain (L-chain).

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 : Extracellular binding domainHN : Translocation domainLC : Enzymatic domain

The Clostridial toxins bind to their target cells via the HCDomain. The HC domain binds to ganglioside targets

Gangliosides are carbohydrate modified sphingolipids foundon the external leaflet of the plasma membrane

Clostridial toxins intracellularise via endosytosis and apH dependent membrane translocation

Mochida et al.,(1990) P.N.A.S.

Injection of light chain mRNA ofClostridial toxins inhibits synaptic transmission.

Clostridial toxin induces Accumulation of synaptic vesiclesat 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 spinalinterneurons

BoTx binds to and is internalisedby motorneurons via endocytosis.The toxin exits from the endosomeand blocks synaptic transmission

TeTx binds to motorneurons, is retrogradely transported along theaxon within a endosomal vesicle. TeTx is then trans-synaptically transported into an inhibitoryInterneuron, 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 theirIntracellular target

How do Clostridial toxins block the synaptic vesicle cycle?

Cleavage of the synapticvesicle protein synaptobrevin/VAMP correlates with tetanusintoxication 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

TM SNARE SNARE

BoTxBoTx

TM SNARE

BoTx

Syntaxin

Synaptobrevin/VAMP

Toxin cleavage sites are preceded by structural motifsnecessary for recognition of target by toxin. SNAREmotifs 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 complexthat is resistant to toxin cleavage.

…and bind two proteins previously identified as essentialto vesicle traffic throughout the cell, NSF and SNAP

NSF is an ATPase

The SNARE proteins plus NSF and SNAP form a 20SComplex.

Importance of SNARE protein integrity for exocytosisin conjunction with the energy input requirement indicates a key point of regulation for vesicle fusion

SNARE proteins have orthologs in yeast that regulateVesicle trafficking fusion steps suggestingthe mechanism of vesicle fusion is ancient (preceeding the evolution of neurotransmission)

Shekman and Novick (2004) Cell 116(suppl)S13-15Novick, 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 hypothesisRothman and Warren (1992) Curr. Biol. 116:135-146

The study of Clostridial toxin action has revealed conservedMechanisms that regulate vesicle fusion and traffic

Clostridial Toxins as tools in cell biology:

Testing of roles of individual SNARE proteins in secretoryprocesses.

Clostridial Toxins as therapeutic tools:

BlepharospasmFacial hemispasmExcessive palm and foot perspirationBotox 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 lobotomyMapping 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 transgenesin 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, 341-351Martin, Keller and Sweeney (2002) Advances in Genetics47, 1-47)

There are two synaptobrevinsIn flies. Only one is cleaved by TeTxLC(Sweeney et al 1995)

Neuronal-TeTxLCMuscle-TeTxLC

Synaptotagminstaining

n-synaptobrevinstaining

Neuronal expression of TeTxLCabolishes n-synaptobrevin staining(Sweeney et al., ‘95)

Neuronal expression of TeTxLC abolishesneuromuscular synaptictransmission(Sweeney et al, ‘95)

Can we use UAS-TeTxLC as a tool to study behaviour?

Tracey et al (2003) Painless,a Drosophila gene essentialfor 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