Two routes to neurotoxicity?

TULLIO POZZAN

BACTERIAL TOXINS

Two routes to neurotoxicity? Tetanus and botulinum toxins cause death by preventing small synaptic vesicle release. Recent results suggest mechanisms for these toxins and may provide new insights into the cell biology of synapses. Bacterial toxins still provide one of the most formidable threats to life. Diphtheria, cholera, tetanus and botulism are the most familiar names of the deadly diseases caused solely by these bacterial weapons. Despite the introduction in the first half of this century of mass vaccination with inactivated toxins, each year bacterial toxins still account for millions oF human deaths, mostly in developing countries, The toxins responsible for the diseases mentioned above act intracellularly and they all consist of two subunits: one subunit binds a receptor on the surface of target cells and translocates the other, catalytically active, subunit into the: cytosol [ 1,2]. Although progress has been made in understanding the catalytic activities of diphtheria and cholera toxins, the catalytic activities of the tetanus (TeTx) and botulinum (BoTx) toxins, made by Clostridium t-etani and Clostridium botulinurn respectively, have been mysterious. At the subcellular level, the effects of TeTx and BoTx are the same - both block exocytosis of small synaptic vesicles As, however, TeTx acts on inhibitory spinal cord interneurons, whereas BoTx acts on spinal cord motorneurons, the two toxins have opposite effects on skeletal muscle, causing spastic and flaccid paralysis, respectively. Determining the molecular mechanisms by which TeTx and BoTx produce their toxic effects is not only important from a medical and social point of view, it is also of interest for basic science as it is likely to increase our understanding of the affected cellular function - neurotransmitter release. Thus, recent results leading to two new suggested mechanisms by which TeTx and BoTx may inhibit small synaptic vesicle exocytosis represent an exciting development in neurobiology [3-51. The new results were (obtained by two groups who both began by analyzing the neurotoxin sequences. Facchiano and Luini noticed that the sequences of both TeTx chains include short stretches with similarities to motifs that occur in the sequences of a number of substrates of cellular transglutaminase [ 31. Transglutaminases are ubiquitous enzymes that catalyse the irreversible crosslinking of proteins [6]. Facchiano and Luini found that TeTx is a stimulater of transglutaminase, increasing the activity of the enzyme, assayed in the presence of physiological Ca2+ and GTP concentrations, more than ten-fold. This appears to be a stoichiometric stimulation, depending on a 1: 1 physic:al interaction between TeTx and transglutaminase. The next step was to identify the important target protein(s) of TeTx-stimulated transglutaminase. Synapsin I, a major protein of synaptic vesicle membranes [7], was Volume 2

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shown to be a preferred substrate of transglutaminase, which in synaptic terminals is localized both in the cy tosol and on synaptic vesicle membranes (Facchiano F, Benfenati F, Valtorta F, Luini A personal communication). Synapsin I is known to provide a dynamic link between synaptic vesicles and the cytoskeleton, and thereby to be an indirect regulator of the interaction between synaptic vesicles and the plasma membrane. Thus, Facchiano et al. suggest that TeTx acts by stimulating transglutaminase, leading to the irreversible, covalent crosslinking of synapsin I to microfilaments. This crosslinking is suggested to ‘freeze’ the synaptic vesicles in the cytosol, preventing them from fusing with the plasma membrane. when Schiavo et al. analysed the neurotoxin sequences, however, they spotted a diierent homology - they noticed that a histidine-rich conserved segment of the TeTx and BoTx light chains contains the sequence His-Glu-X-X-His, where X is any amino acid, characteristic of the E-i”+-binding site of metalloproteases [4,5]. Schiavo et al. then proceeded to test the suggestion that TeTx and BoTx have Zn2+ -dependent protease activity crucial to their toxic effects on neurons. In support of this suggestion they found, first, that highly purified TeTx and BoTx preparations contain Zn2+ ions reversibly bound to their light chains in a 1: 1 stoichiometric ratio. Second, chemical modification and 6?Zn2+ -binding experiments indicated that the Zn2+ atom is indeed coordinated by the histidine-rich segments of the neurotoxins. Third, the authors carried out functional experiments in ~pZysia neurons, a well-characterized model system for studying the intracellular action of TeTx and BoTx, which proved particularly telling. Injection of TeTx or BoTx into 4Zysia neurons inhibits neurotransmitter release, and Schiavo et al. found that with Zn2+-depleted toxins (apotoxins) this effect is greatly delayed. The presence of intracellular heavy-metal ion chelators, or chemical modification of the putative Zn2+ -binding his&dine residues of TeTx and BoTx, were found to prevent completely the in vivo reactivation of the apotoxins. And a known inhibitor of Zn2+ -dependent proteases was found to suppress completely the inhibitory effect of TeTx on neurotransmitter release. If TeTx and BoTx are indeed metalloproteases then presumably they act by cleaving some intracellular protein(s) involved in synaptic vesicle exocytosis. Gel electrophoresis of proteins from highly puriiied synaptic vesicles revealed only one protein, of molecular weight lgk~, cleaved in the presence of the TeTx light chain [4,8]. As expected, only the Zn2+ -containing toxin could cleave the 19 kD protein and cleavage was prevented by Zn2+ 621

protease inhibitors. Imrnunoblot analysis and amino-acid sequencing unambiguously identified the 19kD protein as synaptobrevin 183. Of the two isoforms of synaptobrevin expressed in rat brain [9], only isoform II is cut by TeTx. l?arallel experiments with three BoTx serotypes showed that only BoTx B can cut synaptobrevin II; BoTx A and E are ineffective. This is consistent with electrophysiological evidence that TeTx and BoTx B have the same intracellular target, distinct from that of BoTx A and E. Given that all the BoTx serotypes have the His-Glu-X-X-His motif, however, the prediction is that they should all have Zn2+ -protease activity, but perhaps with different intracellular target(s). The specificity of TeTx and BoTx: B for rat synaptobrevin II is apparently determined by the cleavage site sequence, Gln76-Phe77: in synaptobrevin I position 76 is a Val residue. Injection into Aplysa neurons of a peptide with the sequence spanning the synaptobrevin LI cleavage site (but not the equivalent sequence of synaptobrevin I) was found dras tically to reduce the inhibition of neurotransmitter release by both TeTx and BoTx B. And two known inhibitors of Zn2+-dependent proteases completely blocked the effects of BoTx and TeTx in mice injected with doses of the toxins 1000 times the minimum required for lethality, providing strong evidence that the protease activity is crucial to the in uieto effects of these toxins. The results described above make such a strong case for their being a causal link between the Zn2+ -dependent protease activity of the neurotoxins and the inhibition of neurotransmitter rielease that there seems little room left for the hypothesis put forward by Luini and colleagues.

Fig. 1. Model of the Schiavo et al,

622

mechanism

by which

TeTx and

BoTx

B inhibit

It may turn out, however, that both hypotheses are true. Thus, it remains to be established whether synaptobrevin is the only important target of the protease activity of TeTx and BoTx, and another highly interesting candidate target is transglutaminase itself. As pointed out by Facchiano et al. (personal communication), some transglutaminase isoforms can indeed be activated by km ited proteolysis. A model based on the possibility that TeTx, at least, blocks neurotransmitter release by a dual mechanism - cleavage of synaptobrevin and proteolytic activation of transglutaminase - is depicted in Figure 1. Whether or not transglutaminase activation is rekvant to neurotoxin action, the work of Facchiano et a2. has the important merit of focusing attention on this enzyme, which is abundant in nerve terminals. The location of transglutaminase in nerve terminals and its specific effect on synapsin I suggests the enzyme may have a key role in neurophysiology. Elucidation of the enzy matic activity of neurotoxins, on the other hand, offers new and highly specific tools for neurobiologists, and the existence of drugs that can inhibit the proteolytic activity of the neurotoxins offers for the first time the possibility of curing, rather than just preventing, tetanus and botulism. A number of important basic issues remain unresolved, however. As a cell biologist, three urgent questions come to mind which I expect to be critical to solving the neurosecretion puzzle. First, what proteins, if any, does synaptobrevin interact with on the plasma membrane to allow synaptic vesicle exocytosis. Second, is synaptobrevin merely a docking protein, or does it play

neurotransmitter

release,

based

on the

results

of Facchiano

et al. and

@ 1992 Current Biology

other roles in synaptic vesicle exocytosis? Third, what are the targets of the A and E serotypes of BoTx?

5.

SCHIAVO G, ROSSEITO MONTECUCCO C: Botulinum Biol Chem, in press.

6.

FOLK JE: 49517-531.

7.

VALTORTA F, BENFENATI F, GREENGARD P: Structure and function of the synapsins. J Biol Chem 1992, 267:7195-7198.

8.

SCHIAVO G, BENFENATI F, POULUN B, ROSSE~TO 0, POL~EFUNO DE IAURENTO P, DA~GUPTA BR, MONTECUCCO C: Tetanus and botulinurn-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature, in press.

9.

TRIMEZE WS, COWAN DM, vesicle associated integral

References 1.

At.0~ JE, FREER JH (Ens) Sourcebook London: Academic Press; 1991.

2. SIMPSON LL (ED) San Diego: 3.

4.

Botulinurn

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and

Proteins:

Tetanus Toxin.

1989.

FACCHIANO F, LLIINI A: Tetanus toxin potently stimulates tissue transglutaminase: a possible mechanism of neurotoxicity. J Biol Cbem 1992, 267:132:67-13271. SCHIAVO G, POULAIN B, ROSSETI-o 0, BENFENA~ F, TAUC L, MONTECUCCO C: Tetams toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc. EMBO J 1992, 11:3577-3583.

THE OCTOBER

0,

Transglutaminases.

SANTUCCI neurotoxins Annu

A, DASGUPTA BR, are zinc proteins. J Rev

Biochem

1980,

SCHELIER RH: VAMP-l: a synaptic membrane protein. I+oc Natl Acad

Sci LISA 1988, 85:453%4542.

Tullio Pozzan, Dipartimento di Scienze Biomediche Sperimentali, Universita Degli Studi di Padova, Via Trieste 75, 35121 Padova, Italy. -

1992 ISSUE OF CURRENT OPINION

IN NEUROBIOLOGY

Contained the following reviews on Neuronal and Glial Cell Biology, edited by Regis B. Kelly and Louis F. Reichardt: Neuronal cytoskeleton and growth by Frank Solomon Neuronal polarity by Ann Mane Craig, Mark Jareb and Gary Banker Schwann cells: early lineage, regulation of proliferation and control of myelin formation by Kristjan R. Jessen and Rhona Minsky The transport of neurotransmitters into synaptic vesicles by Robert H. Edwards Phototransduction in DrosopbiZa a paradigm for the genetic dissection of sensory transduction cascades by Charles S. Zuker Reconstituting animals &from immortal precursors by Ron McKay Motors for fast axonal transport by Trina A. Schroer Cell adhesion molecules, second messengers and axonal growth by Patrick Doherty and Frank S. Walsh Membrane trafficking in neurons by Eric Holtzman The same issue also contained the following reviews on Disease, Transplantation and Regeneration, edited by Donald L. Price and Anders Bjdrklund: Cytokines in neural regeneration by Klaus Unsicker, Claudia Grothe, Reiner Westermann and Konstantin Wewetzer Dopaminergic transplants in experimental parkinsonism: cellular meclhanisms of graft-induced functional recovery by Anders Bjijrklund Sodium channel defects in the periodic paralyses by Robert L. Barchi Natural and experimental prion diseases of humans and animals by Stanley B. Prusiner Macrophages and nerve regeneration by V. Hugh Perry and Michael C. Brown Role of excitotoxicity in human neurological disease by M. Flint Beal Amyloidogenesis in Alzheimer’s disease: basic biology and anilnal models by Sangram S. Sisodia and Donald L. Price Retroviruses and nervous system disease by Richard T. Johnson The disordered neuronal cytoskeleton in Alzheimer’s disease by Virginia M-Y. Lee and John Q. Trojanowski

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