NEWS AND COMMENT

Response from Ray

Coffield, Considine and Simpson have reviewed some of the recent developments in understanding the mechanism of action of botulinum toxins, and conclude that one of the proposed mechanisms might lead to the development of a mol- ecular therapy for botulinum toxins. This review is valuable because it summarizes the most recent dis- coveries on the action of botu- linum toxins, all of which have potential importance for the devel- opment of countermeasures against the effects of botulinum toxins.

Coffield et al. have expressed concern about the validity of the proposed mechanism involving the role of phospholipase A, (PLA,) and arachidonic acid in acetyl- choline exocytosis and botulinum A toxin action’. They criticize the fact that data were not presented to support the hypothesis that intra- cellular phospholipase and arachi- donic acid control exocytosis. They also point out that, since all the data were obtained from exper- iments in which drugs were added to the extracellular medium, it is not clear how extracellularly added PLA, or arachidonic acid could permeate through the cell mem- brane to mimic the effects of their acting intracellularly.

I would like to take this oppor- tunity to provide explanations and

-references to other work that may clarify any confusion. There are numerous reports in the literature in which the extracellular addition of PLA, and/or arachidonic acid has been used to study hormone or neurotransmitter release from intra- cellular sites2-‘. Axelrod et aL6 and Lynch and Voss” have suggested that, since it is a small and lipid- soluble molecule, arachidonic acid can cross membranes without dif- ficulty. Bourdeau et al.” and Catania et al.’ suggested that, since exogen- ous PLA, does not cross the plasma membrane, it may act via arachi- donic acid generated by the action of PLA, on membrane phospho- lipids.

To examine the role of PLAz in exocytosis, we used the bee-venom I’LA? activator, melittin, which is a hydrophobic peptide that has been

proposed to stimulate phospho- lipases localized both outside and inside the cell membrane8. Melittin itself has not been reported to have any phospholipase or membrane- lytic activity. We observed that 1 pM melittin added to the extracellu- lar medium markedly augmented LiHlacetylcholine release caused by the submaximal stimulation of PC12 cells with 40mM KCI. These results suggest a specific role for PLA, in acetylcholine exocytosis, involving PLA,-induced arachi- donic acid release from membrane phospholipids localized on the ex- ternal and/or internal aspects of the membrane. Mayorga et aL9 have emphasized that arachidonic acid is required specifically, as well as other known fusion factors, for the membrane fusion event itself. Like Coffield et al., we were also concerned about whether exogen- ously added arachidonic acid would penetrate the membrane to act inside the cell as a fusogen. We ob- served that exogenous arachidonic acid, PLA, or the PLA, activator melittin increased basal (unstimu- lated) [‘H jacetylcholine release slightly (by approx. 10%). How- ever, both arachidonic acid and mel- ittin greatly augmented the release when the membrane was depolar- ized by the addition of 40 mM KCl. These results suggest that (1) these agents are not acting via nonspecific membrane effects, such as leakage, and (2) membrane depolarization is necessary for exogenous arachi- donic acid to facilitate exocytosis.

We propose that exogenous arachi- donic acid may be transported across the membrane under depolar- izing conditions, as has also been shown by other laboratoriesiO~“. Further studies are in progress in our laboratory to explain the de- tailed mechanism of depolarization- induced arachidonic acid transport across the cell membrane.

Prabhati Ray Dept of Biology, Divn of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington DC 20067, USA

References 1 Ray, P. et al. (1993) J. Biol. Chem. 268,

11057-11064 2 Badi, A. et al. (1986) Endocrinology 119,

1427-1431 3 Lynch, M.A. and Voss, K.L. (1990)

J. Neurochem. 55,215-221 4 Lafrance, M. and Hansel, W. (1992)

Proc. Sot. Exp. Biol. Med. 201, 106-113

5 Bourdeau, A. et al. (1992) Endocrinology 130,1339-1344

6 Axelrod, J., Burch, R.M. and Jelsema, C.L. (1988) Trends Neurosci. 11, 117-123

7 Catania, M.V. et al. (1993) 1. Neurochem. 60,236-245

8 Mollay, C. and Kreil, G. (1974) FEBS Lett. 46, 141-144

9 Mayorga, L.S. et al. (1993) Proc. Nut! Acud. Sci. USA 90,10255-10259

10 Kelleher, J.A. and Sun, G.Y. (1985) Neurochem. lnt. 7, 825-831

11 Dorman, R.V., Schwartz, M.A. and Terrian, D.M. (1988) Brain Res. Bull. 21,445-450

Response from Facchiano, lnnamorati and Luini

Coffield et al. discuss three hypoth- eses that have been proposed re- cently for the mode of action of the clostridial neurotoxins. They con- clude that the mechanism of toxin action suggested by Schiavo et al. ‘v2, that is, by proteolytic cleavage of essential synaptic proteins, is sup- ported by substantial evidence and explains toxin-induced neuropar- alysis elegantly. By contrast, they suggest that there are reasons to be cautious about the other two

hypotheses, which implicate the en- zymes transglutaminase (TGase)“,4 and phospholipase A, (PLA,)’ in the neuroparalytic effects of the toxins.

We could not agree more about the power and elegance of the pro- teolytic mechanism first demon- strated by Cesare Montecucco and colleagues, and later confirmed by Link et aL6. Indeed, we believe that their idea that the short-term effects of clostridial toxins depend on the proteolysis of proteins essential for exocytosis’ is sound, and will be rapidly and universally accepted. Moreover, we agree that we should

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NEWS AND COMMENTI

be cautious about the TGase hy- pothesis (which we are specifi- cally concerned with in this letter), although not for the reasons dis- cussed by Coffield et al., which appear to us to be rather weak. The TGase hypothesis proposes that the light chain of tetanus toxin (Tetox) acts by activating an intrasynaptic TGase, resulting in crosslinking and inactivation of synapsin I, an excel- lent TGase substrate and a protein that is central to the regulation of exocytosis in the brain8.

Coffield et al. note that there is no compelling evidence that TGase is involved in neurosecretion. But this is not a logical obstacle to think- ing that Tetox might act by acti- vating TGase; indeed, one of the main goals in Tetox research has tra- ditionally been to reveal the involve- ment of novel or known proteins in exocytosis. Coffield et al. note that the concentrations of Tetox that stimulate TGase (nM) are too high to be functionally relevant in viuo. However, since the maximally active concentrations of Tetox are stoichiometrically related to the con- centrations of TGase (nM) present in in vitro experiments (an obser- vation Coffield et al. find curious), then the concentrations of Tetox that would be expected to stimulate TGase must be in same range (nM), even though the affinity between the two proteins is likely to be very high. Finally, Coffield et al. re- mark that there is no evidence that Tetox stimulates TGase in intact cells; interestingly, however, we have found recently that Tetox intoxication is associated with a marked increase in TGase-depen- dent protein crosslinking in isolated nerve endings (F. Facchiano et al., unpublished).

The main reason why we think that we should be cautious about the TGase hypothesis is, quite simply, that we have been unable so far to confirm or reject it experimentally, and that, unfortunately, the lack of potent and specific inhibitors of TGase makes it difficult to ob- tain conclusive results. The second reason is economy of thought: why should we speculate about the in- volvement of TGase in Tetox action if another simple and elegant ex- planation (synaptobrevin cleavage)

is already at hand? We feel that this might be, understandably, an under- lying cause for the reservations Coffield et al. seem to have about the TGase hypothesis. However, in biology, simplicity and elegance of reasoning, so effective in other fields of research such as physics, might lead one to overlook signifi- cant facts. Clostridial intoxication is nearly irreversible. It is entirely possible that, while the rapid phase (minutes to hours) of toxin action could be mediated by the proteo- lytic mechanism revealed by Schiavo et al., the longer-term effects (days) might be sustained by the slow accumulation of crosslinked prod- ucts of Tetox-activated synaptic TGase. Redundancy of biological mechanisms is so common that this scenario would not be at all sur- prising. By the same argument, we feel that the involvement of PLA, in the action of botulinum neuro- toxin type A should not be dis- counted. Clostridial toxins might still offer useful clues towards clari- fying the regulatory mechanisms of exocytosis.

Acknowledgements The authors are supported by the Agenzia per la Promozione dello Sviluppo nel Mezzogiorno PR-2 and the Italian National Research Council (Convenzione CNR-Consorzio Mario Negri Sud).

Francesco Facchiano, Giulio Innamorati and Albert0 Luini Laboratory of Molecular Neurobiology, Mario Negri Sud Institute, 66030 S. Maria Imbaro (Chieti), Italy

References 1 Schiavo, G. et al. (1992) EMBO 1. 11,

3577-3583 2 Schiavo, G. et al. (1992) Nature 359,

832-835 3 Facchiano, F. and Luini, A. (1992)

J. Biol. Chem. 267,13267-13271 4 Facchiano, F. et al. (1993) /. Biol. Chem.

268,4588-4591 5 Ray, P. et al. (1993) J. Biol. Chem. 268,

11057-11064 6 Link, E. et al. (1992) Biochem. Biophys.

Res. Commtm. 189,1017-1023 7 Montecucco, C. and Schiavo, G. (1993)

Trends Biochem. Sci. 18324-327 8 Facchiano, F. et al. (1993) Trends

Biochem. Sci. 1X,327-329

Comment from Coffield, Considine and Simpson

We thank Schiavo and Montecucco, Ray, and Facchiano, Innamorati and Luini for their responses to our article. In each case, the responses raise interesting points.

We agree almost completely with the comments by Schiavo and Montecucco. To the extent that there is any difference between our opinions, it relates mainly to levels of optimism. Schiavo and Montecucco believe that it may be possible to identify or synthesize a protease inhibitor that will diffuse across neuronal membranes and antagon- ize the actions of intracellular toxin. We wonder whether the likelihood of success would be enhanced by identifying not only an appropri- ate inhibitor, but also an efficient technique for targeting the inhibi- tor to the tissue. In this particular case, we would be perfectly happy if the optimism of Schiavo and Montecucco is warranted, because

this would almost certainly ensure that a therapeutic agent will be rapidly discovered.

We have carefully examined the comments by Ray and, with due respect, we must note that they do not persuade us to change our opinion. We are familiar with the references cited by Ray, but none of them demonstrates compellingly that phospholipase-AZ-generated arachidonic acid triggers fusion and exocytosis. This reservation certainly applies to the neuromus- cular junction (and see below). We must also repeat that it is difficult to see how extracellular phos- pholipase and/or arachidonic acid can mimic the putative intracellular actions of these agents in stimu- lating the release of acetylcholine. Exocytosis of transmitter is a regulated event, not a constitutive one. Virtually all research on neurotransmitter release envisions the process as having a rapid sequence of signal-mediated events. The slow and constant flux of an

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