Advertisement

Evaluation of the Therapeutic Usefulness of Botulinum Neurotoxin B, C1, E, and F Compared with the Long Lasting Type A

BASIS FOR DISTINCT DURATIONS OF INHIBITION OF EXOCYTOSIS IN CENTRAL NEURONS*
Open AccessPublished:October 14, 2002DOI:https://doi.org/10.1074/jbc.M209821200
      Seven types (A–G) of botulinum neurotoxin (BoNT) target peripheral cholinergic neurons where they selectively proteolyze SNAP-25 (BoNT/A, BoNT/C1, and BoNT/E), syntaxin1 (BoNT/C1), and synaptobrevin (BoNT/B, BoNT/D, BoNT/F, and BoNT/G), SNARE proteins responsible for transmitter release, to cause neuromuscular paralysis but of different durations. BoNT/A paralysis lasts longest (4–6 months) in humans, hence its widespread clinical use for the treatment of dystonias. Molecular mechanisms underlying these distinct inhibitory patterns were deciphered in rat cerebellar neurons by quantifying the half-life of the effect of each toxin, the speed of replenishment of their substrates, and the degradation of the cleaved products, experiments not readily feasible at motor nerve endings. Correlation of target cleavage with blockade of transmitter release yielded half-lives of inhibition for BoNT/A, BoNT/C1, BoNT/B, BoNT/F, and BoNT/E (≫31, ≫25, ∼10, ∼2, and ∼0.8 days, respectively), equivalent to the neuromuscular paralysis times found in mice, with recovery of release coinciding with reappearance of the intact SNAREs. A limiting factor for the short neuroparalytic durations of BoNT/F and BoNT/E is the replenishment of synaptobrevin or SNAP-25, whereas pulse labeling revealed that extended inhibition by BoNT/A, BoNT/B, or BoNT/C1 results from longevity of each protease. These novel findings could aid development of new toxin therapies for patients resistant to BoNT/A and effective treatments for human botulism.
      BoNT
      botulinum neurotoxin
      DIV
      days in vitro
      KRH
      Krebs-Ringer-HEPES
      LC
      light chain
      Sbr
      synaptobrevin
      SNAP-25
      25-kDa synaptosomal-associated protein
      SNARE
      solubleN-ethylmaleimide-sensitive factorattachment protein receptor
      STx1
      syntaxin1
      TeTx
      tetanus toxin
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      Seven immunologically distinct serotypes of botulinum neurotoxin (BoNT)1 (A–G) fromClostridium botulinum are homologous proteins consisting of a heavy and light chain linked by an essential disulfide and noncovalent interactions that specifically block the release of acetylcholine at the neuromuscular junction (reviewed in Refs.
      • Cherington M.
      ,
      • Dolly J.O.
      • Lisk G.
      • Foran P.G.
      • Meunier F.
      • Mohammed N.
      • O'Sullivan G.
      • dePaiva A.
      ,
      • Schiavo G.
      • Matteoli M.
      • Montecucco C.
      ). BoNTs cause botulism, the majority of human outbreaks being caused by types A, B, or E (
      • Cherington M.
      ); however, they are remarkably useful as therapeutic agents (see below). The striking potency of the toxins and their cholinergic selectivity arise from their multiple domains mediating: (i) targeting to motor nerve endings via high affinity interaction with ecto-acceptors located exclusively thereon (
      • Dolly J.O.
      • Black J.
      • Williams R.S.
      • Melling J.
      ,
      • Daniels-Holgate P.U.
      • Dolly J.O.
      ) and (ii) endocytosis (
      • Black J.D.
      • Dolly J.O.
      ) followed by translocation of a LC-containing moiety into the cytosol. Their LCs are Zn2+-dependent endoproteases that selectively cleave single peptide bonds (except for BoNT/C1; see below) in one of three SNARE proteins that constitute the components of a ternary complex responsible for vesicle docking/fusion during regulated exocytosis (
      • Sollner T.
      • Bennett M.K.
      • Whiteheart S.W.
      • Scheller R.H.
      • Rothman J.E.
      ). Synaptosomal-associated protein of 25 kDa (SNAP-25) (
      • Oyler G.A.
      • Higgins G.A.
      • Hart R.A.
      • Battenberg E.
      • Billingsley M.
      • Bloom F.E.
      • Wilson M.C.
      ) is proteolyzed by BoNT/A, BoNT/C1, and BoNT/E at separate sites near the C terminus: Gln197–Arg198, Arg198–Ala199, and Arg180–Ile181, respectively (
      • Schiavo G.
      • Matteoli M.
      • Montecucco C.
      ). Another plasmalemmal protein, syntaxin1 (STx1) (reviewed in Ref.
      • Bennett M.K.
      • Garcia-Arraras J.E.
      • Elferink L.A.
      • Peterson K.
      • Fleming A.M.
      • Hazuka C.D.
      • Scheller R.H.
      ), is also cleaved by BoNT/C1, and synaptobrevin, a synaptic vesicle protein (Sbr) (
      • Trimble W.S.
      • Cowan D.M.
      • Scheller R.H.
      ,
      • Baumert M.
      • Maycox P.R.
      • Navone F., De
      • Camilli P.
      • Jahn R.
      ) is cleaved by BoNT/B, BoNT/D, BoNT/F, BoNT/G, and tetanus toxin (TeTx). BoNT/A- or BoNT/E-truncated SNAP-25 (termed SNAP-25A or SNAP-25E, respectively) remains membrane-bound, but release is inhibited; in the case of SNAP-25A, some assembly and disassembly of the ternary complex can still occur (
      • Hayashi T.
      • McMahon H.
      • Yamasaki S.
      • Binz T.
      • Hata Y.
      • Südhof T.C.
      • Niemann H.
      ,
      • Pellegrini L.L.
      • O'Connor V.
      • Lottspeich F.
      • Betz H.
      ). Truncation of STx1 or Sbr by the requisite BoNT results in detachment of their cytosolic domains.
      When applied locally to humans for the treatment of dystonias (reviewed in Ref.
      • Brin M.F.
      ), BoNT/A, BoNT/B, and BoNT/E cause neuromuscular paralysis for more than 4 months, ∼2 months, or <4 weeks, respectively (
      • Sloop R.R.
      • Cole B.A.
      • Escutin R.O.
      ,
      • Eleopra R.
      • Tugnoli V.
      • Rossetto O., De
      • Grandis D.
      • Montecucco C.
      ); the limited results available for type C1 suggest a duration less than or equal to that of BoNT/A (
      • Eleopra R.
      • Tugnoli V.
      • Rossetto O.
      • Montecucco C.
      • De Grandis D.
      ). It is unclear why the recovery times in rodents are shorter and yet show the same rank order (1–2 months (BoNT/A), 21 days (BoNT/B), 7 days (BoNT/F), and 4 days (BoNT/E)) (
      • de Paiva A.
      • Meunier F.A.
      • Molgó J.
      • Aoki K.R.
      • Dolly J.O.
      ,
      • Jurasinski C.V.
      • Lieth E.
      • Dang Do A.N.
      • Schengrund C.L.
      ).
      Meunier, F. A., Lisk, G., Sesardic, D., and Dolly, J. O. (2003) Mol. Cell. Neurobiol., in press.
      2Meunier, F. A., Lisk, G., Sesardic, D., and Dolly, J. O. (2003) Mol. Cell. Neurobiol., in press.
      Insight has been gained into the sequence of events involved in the protracted resumption of neurotransmission in BoNT/A-poisoned motor endplates by monitoring synaptic function in individually identified nerve endings of living mice (
      • de Paiva A.
      • Meunier F.A.
      • Molgó J.
      • Aoki K.R.
      • Dolly J.O.
      ). This showed that the transient appearance of functional nerve sprouts mediates a partial return of neuromuscular function, with full recovery relying on the originally affected endings reacquiring the ability to mediate chemical transmission. In chromaffin cells, the persistence of BoNT/A protease for many weeks contributes to the extended inhibition of secretion; also, SNAP-25A has been shown to be inhibitory (
      • Huang X.H.
      • Wheeler M.B.
      • Kang Y.H.
      • Sheu L.
      • Lukacs G.L.
      • Trimble W.S.
      • Gaisano H.Y.
      ,
      • O'Sullivan G.A.
      • Mohammed N.
      • Foran P.G.
      • Lawrence G.W.
      • Dolly J.O.
      ,
      • Criado M.
      • Gil A.
      • Viniegra S.
      • Gutierrez L.M.
      ). Of particular interest, Eleopraet al. (
      • Eleopra R.
      • Tugnoli V.
      • Rossetto O., De
      • Grandis D.
      • Montecucco C.
      ) reported that co-treating human endplates with BoNT/A and BoNT/E results in a more rapid recovery of neuromuscular function, equivalent to that of BoNT/E alone, prompting many scientists (
      • Eleopra R.
      • Tugnoli V.
      • Rossetto O., De
      • Grandis D.
      • Montecucco C.
      ,
      • O'Sullivan G.A.
      • Mohammed N.
      • Foran P.G.
      • Lawrence G.W.
      • Dolly J.O.
      ,
      • Raciborska D.A.
      • Charlton M.P.
      )2 to suggest that both proteases have equivalent lifetimes in the motor nerve ending and the prolonged paralysis by BoNT/A arises from slow replacement of SNAP-25A. Accordingly, Meunier et al. 2 observed that type E hastens the removal of inhibitory SNAP-25A from BoNT/A-treated mouse neuromuscular synapses by converting it to SNAP-25E, which is replaced rapidly; thus, resumption of synaptic transmission is accelerated.
      In this study, biochemical analyses (not practical with motor nerve endings or isolated motoneurons; see “Discussion”) were performed on cultured cerebellar neurons to quantify the half-lives of toxin inhibition and the rates of turnover of SNAREs and their toxin-cleaved products. Although noncholinergic, these neurons provide a useful model for studying the intracellular fate of BoNTs, because we observed the same relative durations of neuroparalytic actions of BoNT/A, BoNT/B, BoNT/C1, BoNT/E, and BoNT/F as measured in motor nerves in vivo (see above). In addition, these homogeneous cerebellar neurons are very susceptible to BoNTs and could be obtained in sufficient numbers for these quantitative measurements. In this way, we have extended earlier findings (
      • O'Sullivan G.A.
      • Mohammed N.
      • Foran P.G.
      • Lawrence G.W.
      • Dolly J.O.
      ,
      • Keller J.E.
      • Neale E.A.
      • Oyler G.
      • Adler M.
      ) and explained how exocytosis can be blocked for dissimilar periods by the different BoNT serotypes.

      DISCUSSION

      The detailed pulse-chase study of native and BoNT-cleaved SNAREs reported herein provides the first unambiguous and direct demonstration of a persistence of BoNT/A protease in central neurons, together with convincing evidence that it is the major factor responsible for prolonged inhibition of neuroexocytosis. Unexpectedly, SNAP-25A exhibited the same turnover rate as the full-sized protein in cerebellar neurons, in contrast with its reported persistence (
      • Dolly J.O.
      • Lisk G.
      • Foran P.G.
      • Meunier F.
      • Mohammed N.
      • O'Sullivan G.
      • dePaiva A.
      ,
      • Raciborska D.A.
      • Charlton M.P.
      )2 in peripheral motor nerve endings. Apparently, an exceptional situation must exist in motor nerve terminals in vivo (discussed in Refs.
      • Dolly J.O.
      • Lisk G.
      • Foran P.G.
      • Meunier F.
      • Mohammed N.
      • O'Sullivan G.
      • dePaiva A.
      and
      • O'Sullivan G.A.
      • Mohammed N.
      • Foran P.G.
      • Lawrence G.W.
      • Dolly J.O.
      ),2 allowing SNAP-25A to squat at the presynaptic membrane because co-treatment of human or murine endplates with BoNT/A and BoNT/E causes a rapid recovery, equivalent to that of BoNT/E alone (
      • Eleopra R.
      • Tugnoli V.
      • Rossetto O., De
      • Grandis D.
      • Montecucco C.
      ).2 The latter would seem to exclude an adequate level of toxin protease persisting—but another study did not detect such a rescue although different conditions (e.g.higher toxin dose) were used (
      • Adler M.
      • Keller J.E.
      • Sheridan R.E.
      • Deshpande S.S.
      )—though perturbation of the otherwise persistant BoNT/A protease activity or localization following treatment with BoNT/E cannot be precluded. Notably, BoNT/A protease persisted unabated for longer than 1 month in cerebellar neurons, thereby precluding BoNT/E-mediated rescue of exocytosis or depletion of SNAP-25A; the apparent lack of replacement of the latter has been observed previously for spinal cord neurons in culture, although SNAP-25 turnover or protease longevity were not directly measured (
      • Keller J.E.
      • Neale E.A.
      • Oyler G.
      • Adler M.
      ). Similarly, a study performed on cultured neuroendocrine cells observed negligible recovery of catecholamine release or replacement of SNAP-25A over 2 months following BoNT/A treatment, apparently resulting from protease persistence (
      • O'Sullivan G.A.
      • Mohammed N.
      • Foran P.G.
      • Lawrence G.W.
      • Dolly J.O.
      ). Therefore, SNAP-25A, but not the E-truncated protein, is retained in motor nerve terminals in vivo at the synaptic vesicle release sites; this intriguing dissimilarity with peripheral and central neurons in vitro warrants further investigation.
      Despite the obvious differences that exist between central cerebellar neurons and motor nerves, many similar neuronal characteristics are conserved; these include common exocytotic mechanisms and proteins, neurite extension, and synapse development. Also, our data reveal that picomolar concentrations of several BoNT serotypes block exocytosis when directly applied to central neurons in culture with potencies matching that observed for motor nerve terminals. In vivo, this has not been observed because toxin access to central and nonmotor spinal neurons is largely prevented by anatomical barriers (e.g. the blood brain barrier). Moreover, BoNTs do not exhibit detectable levels of retrograde transport, characteristic of TeTx. Preliminary unpublished studies comparing BoNT potency in cultured central neurons and motoneurons have indicated that BoNTs poison cholinergic nerves more rapidly. However, if toxin exposures are performed overnight (i.e. when the rate of toxin internalization is not the limiting factor), comparable potencies were observed in both cell types. Most importantly, however, for the purpose of this study concerned with the bases for the different longevities of BoNT serotypes, their relative lifetimes in these neurons are remarkably similar to the distinct durations of neuromuscular paralysis observed in vivo for rodents (see the Introduction).
      Generation of an avid antibody specific for the LC protease of BoNT/E has allowed tracking of the minute quantities that remain after exposure to nanomolar concentrations. Immunoblotting of cell extracts, after a 2-h treatment with BoNT/E, for several chase periods up to 3 days later revealed that the majority of BoNT/E LC remained as a covalently linked di-chain, inconsistent with its delivery to the cytosol (where it would have been reduced). Therefore, there are at least two pools of toxin in these neurons: endosomal and cytosolic. Although it was necessary to use concentrations of toxins supermaximal to those needed to inhibit exocytosis, nevertheless, thet 12INH values shown herein correspond to a t 12 of ∼16 h obtained for cell-associated BoNT/E LC immunoreactivity (data not shown).
      The different degradation rates found herein for SNAP-25 in developing and mature cerebellar granule neurons (∼1 and 2 days, respectively) accord with data from earlier studies (
      • Sanders J.D.
      • Yang Y.
      • Liu Y.
      ), which showed that the accumulation of SNAP-25 during development of neurons results from both increased expression and reduced rates of degradation, processes that stabilize by 14 DIV. The t 12 values ofSbr2 and STx1 in mature neurons (∼4.5 and ∼6 days) are reported for the first time. These collective findings allowed consideration of the contribution that toxin-truncated SNARE replacement makes to the different durations of transmitter release inhibition by BoNT serotypes. Indeed, the results suggest that the rate of SNAP-25 synthesis governs the length of BoNT/E-induced inhibition. Interestingly, removal of up to 26 C-terminal residues from SNAP-25 does not alter its degradation rate, implicating other signals for regulation of its turnover. The rates of synthesis and degradation ofSbr2 must be more rapid in developing neurons relative to the much longer t 12 of 4–5 days observed for the fully mature protein (i.e. analogous to SNAP-25), because at 12INH of ∼ 2 days was found for BoNT/F in developing neurons. Because another Sbr-cleaving toxin, BoNT/B, persists for much longer (t 12INH = ∼10 days) than the periods required for SNARE synthesis or degradation of the truncated N-terminal fragment, persistence of its protease must account for the prolonged inhibition of exocytosis.
      Recent work (
      • Aoki K.R.
      ) highlighted the potential risks associated with the clinical use of large quantities of BoNT/B for achieving paralysis of medium length, because of a much reduced safety margin relative to BoNT/A. Although the t 12INH values determined herein are dependent upon both the times required for removal of the BoNT protease and replacement of cleaved SNARE with intact, protease persistence primarily dictates the largert 12INH values measured in neurons treated with BoNT/A, BoNT/C1, or BoNT/B. Attempts by others to examine the t 12 of the LC of the closely related Clostridial neurotoxin, TeTx, in cultured spinal neurons, found that a highly radio labeled toxin disappeared long before even an initial onset of recovery from blockade of neurotransmission (
      • Habig W.H.
      • Bigalke H.
      • Bergey G.K.
      • Neale E.A.
      • Hardegree M.C.
      • Nelson P.G.
      ); the authors correctly suggest that degradation of TeTx LC (t 12= ∼6 days) may underlie the slow recovery from neuroinhibition. Indeed, it has been estimated that only 10–100 intracellular toxin molecules are required to inhibit exocytosis (
      • Erdal E.
      • Bartels F.
      • Binscheck T.
      • Erdmann G.
      • Frevert J.
      • Kistner A.
      • Weller U.
      • Wever J.
      • Bigalke H.
      ), precluding straightforward radiolabeled detection; furthermore, this approach does not distinguish between relevant functional toxin protease in the cytosol and that which may reside in other cellular locations (i.e. endosomes). Therefore, the methodology used herein for measuring the kinetics of recovery from inhibition offers obvious advantages.
      Detailed BoNT dose dependence studies revealed good correlations between losses of intact SNAREs and inhibition of evoked transmitter release, providing a direct demonstration of their involvement in up to 90% of the Ca2+-dependent evoked glutamate exocytosis measured. Note that microanatomical features of motor neurons in vivoare not reproduced by neurons in culture (including motoneurons), and they could play important roles in determining the duration, localization, and molecular basis of paralysis (
      • Dolly J.O.
      • Lisk G.
      • Foran P.G.
      • Meunier F.
      • Mohammed N.
      • O'Sullivan G.
      • dePaiva A.
      ). However, an imperfect relationship was observed regarding SNAP-25A content and inhibition of evoked release in BoNT/A-treated cells; this component of release (∼30% of the total) is apparently mediated by SNAP-25A because it was reduced by sequential BoNT/E administration. A similar situation has been found in permeabilized neuroendocrine cells (
      • Lawrence G.W.
      • Foran P.
      • Mohammed N.
      • DasGupta B.R.
      • Dolly J.O.
      ,
      • Dolly J.O.
      • Lawrence G.W.
      • Foran P.
      ) and synaptosomes (
      • Ashton A.C.
      • Dolly J.O.
      ).
      A small number of patients are primary nonresponders to BoNT/A therapy; also, multiple administrations may gradually elicit immunity in a tiny minority of responders and limit the efficacy of treatment (reviewed in Ref.
      • Brin M.F.
      ). Therefore, an alternative serotype with the potency and duration of type A is required. In this context, these studies have demonstrated that BoNT/C1 may possess such therapeutic potential (
      • Eleopra R.
      • Tugnoli V.
      • Rossetto O.
      • Montecucco C.
      • De Grandis D.
      ), except that it has been reported to impair neurite/axonal growth and cause cell death, an effect not ascribable to contamination (Ref.
      • Williamson L.C.
      • Neale E.A.
      and this work). From the present investigation, it seems that such BoNT/C1 toxicity may result from its proteolysis of STx1 because the dose dependence study revealed that only minimal cleavage of STx1 coincides with the lethal effects, whereas extensive SNAP-25 cleavage was not lethal; also, the SNAP-251–198 fragment is known to be nonlethal (
      • O'Sullivan G.A.
      • Mohammed N.
      • Foran P.G.
      • Lawrence G.W.
      • Dolly J.O.
      ). Additional proteolysis of one or more of the other five syntaxin isoforms reported (
      • Bennett M.K.
      • Garcia-Arraras J.E.
      • Elferink L.A.
      • Peterson K.
      • Fleming A.M.
      • Hazuka C.D.
      • Scheller R.H.
      ) has not been excluded; only STx4 and STx5 are known to be resistant to BoNT/C1 (reviewed in Ref.
      • Schiavo G.
      • Matteoli M.
      • Montecucco C.
      ). An essential nonsynaptic vesicle docking fusion role for STx1 in developing neurons is suggested by its notable abundance in immature cerebellar neurons, which are almost devoid of the other SNAREs and lack the functional Ca2+-dependent exocytotic machinery (Fig. 1 C). In conclusion, this first detailed examination of the molecular basis for the extended action of BoNT/A relative to shorter acting serotypes in neurons has provided novel information that should aid the extension of therapies as well as the development of countermeasures for botulism.

      Acknowledgments

      We thank M. C. Goodnough, W. H. Tepp, and C. J. Molizio for purifying BoNT/B and/E in the laboratory of E. A. Johnson.

      REFERENCES

        • Cherington M.
        Muscle Nerve. 1998; 21: 701-710
        • Dolly J.O.
        • Lisk G.
        • Foran P.G.
        • Meunier F.
        • Mohammed N.
        • O'Sullivan G.
        • dePaiva A.
        Brin M. Jankovic J. Hallet M. Scientific and Therapeutic Aspects of Botulinum Toxins. Lippincott Williams and Wilkins, Philadelphia, PA2002: 91-102
        • Schiavo G.
        • Matteoli M.
        • Montecucco C.
        Physiol. Rev. 2000; 80: 717-766
        • Dolly J.O.
        • Black J.
        • Williams R.S.
        • Melling J.
        Nature. 1984; 307: 457-460
        • Daniels-Holgate P.U.
        • Dolly J.O.
        J. Neurosci. Res. 1996; 44: 263-271
        • Black J.D.
        • Dolly J.O.
        J. Cell Biol. 1986; 103: 534-544
        • Sollner T.
        • Bennett M.K.
        • Whiteheart S.W.
        • Scheller R.H.
        • Rothman J.E.
        Cell. 1993; 75: 409-418
        • Oyler G.A.
        • Higgins G.A.
        • Hart R.A.
        • Battenberg E.
        • Billingsley M.
        • Bloom F.E.
        • Wilson M.C.
        J. Cell Biol. 1989; 109: 3039-3052
        • Bennett M.K.
        • Garcia-Arraras J.E.
        • Elferink L.A.
        • Peterson K.
        • Fleming A.M.
        • Hazuka C.D.
        • Scheller R.H.
        Cell. 1993; 74: 863-873
        • Trimble W.S.
        • Cowan D.M.
        • Scheller R.H.
        Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4538-4542
        • Baumert M.
        • Maycox P.R.
        • Navone F., De
        • Camilli P.
        • Jahn R.
        EMBO J. 1989; 8: 379-384
        • Hayashi T.
        • McMahon H.
        • Yamasaki S.
        • Binz T.
        • Hata Y.
        • Südhof T.C.
        • Niemann H.
        EMBO J. 1994; 13: 5051-5061
        • Pellegrini L.L.
        • O'Connor V.
        • Lottspeich F.
        • Betz H.
        EMBO J. 1995; 14: 4705-4713
        • Brin M.F.
        Muscle Nerve. 1997; S6 (suppl.): 146-168
        • Sloop R.R.
        • Cole B.A.
        • Escutin R.O.
        Neurology. 1997; 49: 189-194
        • Eleopra R.
        • Tugnoli V.
        • Rossetto O., De
        • Grandis D.
        • Montecucco C.
        Neurosci. Lett. 1998; 256: 135-138
        • Eleopra R.
        • Tugnoli V.
        • Rossetto O.
        • Montecucco C.
        • De Grandis D.
        Neurosci. Lett. 1997; 224: 91-94
        • de Paiva A.
        • Meunier F.A.
        • Molgó J.
        • Aoki K.R.
        • Dolly J.O.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3200-3205
        • Jurasinski C.V.
        • Lieth E.
        • Dang Do A.N.
        • Schengrund C.L.
        Toxicon. 2001; 39: 1309-1315
        • Williamson L.C.
        • Neale E.A.
        J. Neurosci. Res. 1998; 52: 569-583
        • Huang X.H.
        • Wheeler M.B.
        • Kang Y.H.
        • Sheu L.
        • Lukacs G.L.
        • Trimble W.S.
        • Gaisano H.Y.
        Mol. Endocrin. 1998; 12: 1060-1070
        • O'Sullivan G.A.
        • Mohammed N.
        • Foran P.G.
        • Lawrence G.W.
        • Dolly J.O.
        J. Biol. Chem. 1999; 274: 36897-36904
        • Criado M.
        • Gil A.
        • Viniegra S.
        • Gutierrez L.M.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261
        • Raciborska D.A.
        • Charlton M.P.
        Can. J. Physiol. Pharmacol. 1999; 77: 679-688
        • Keller J.E.
        • Neale E.A.
        • Oyler G.
        • Adler M.
        FEBS Letts. 1999; 456: 137-142
        • Lawrence G.W.
        • Foran P.
        • Dolly J.O.
        Eur. J. Biochem. 1996; 236: 877-886
        • Chen F.S.
        • Foran P.
        • Shone C.C.
        • Foster K.A.
        • Melling J.
        • Dolly J.O.
        Biochemistry. 1997; 36: 5719-5728
        • Cambray-Deakin M.A.
        Cohen J. Wilkin G.P. Neural Cell Culture: A Practical Approach. IRL Press, Oxford, UK1995: 3-13
        • Thangnipon W.
        • Kingsbury A.
        • Webb M.
        • Balazs R.
        Brain Res. 1983; 313: 177-189
        • Kingsbury A.E.
        • Gallo V.
        • Woodhams P.L.
        • Balazs R.
        Brain Res. 1985; 349: 17-25
        • Gallo V.
        • Ciotti M.T.
        • Coletti A.
        • Aloisi F.
        • Levi G.
        Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7919-7923
        • Schramm M.
        • Eimerl S.
        • Costa E.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1193-1197
        • Gallo V.
        • Kingsbury A.
        • Balazs R.
        • Jorgensen O.S.
        J. Neurosci. 1987; 7: 2203-2213
        • Van Vliet B.J.
        • Sebben M.
        • Dumuis A.
        • Gabrion J.
        • Bockaert J.
        • Pin J.P.
        J. Neurochem. 1989; 52: 1229-1239
        • Cousin M.A.
        • Nicholls D.G.
        J. Neurochem. 1997; 69: 1927-1935
        • Ravichandran V.
        • Chawla A.
        • Roche P.A.
        J. Biol. Chem. 1996; 271: 13300-13303
        • Foran P.G.P.
        • Fletcher L.M.
        • Oatey P.B.
        • Mohammed N.
        • Dolly J.O.
        • Tavare J.M.
        J. Biol. Chem. 1999; 274: 28087-28095
        • Schiavo G.
        • Benfenati F.
        • Poulain B.
        • Rossetto O.
        • Delaureto P.P.
        • DasGupta B.R.
        • Montecucco C.
        Nature. 1992; 359: 832-835
        • Lawrence G.W.
        • Foran P.
        • Mohammed N.
        • DasGupta B.R.
        • Dolly J.O.
        Biochemistry. 1997; 36: 3061-3067
        • Foran P.
        • Lawrence G.
        • Dolly J.O.
        Biochemistry. 1995; 34: 5494-5503
        • Adler M.
        • Keller J.E.
        • Sheridan R.E.
        • Deshpande S.S.
        Toxicon. 2001; 39: 233-243
        • Sanders J.D.
        • Yang Y.
        • Liu Y.
        J. Neurosci. Res. 1998; 53: 670-676
        • Aoki K.R.
        Toxicon. 2002; 40: 923-928
        • Habig W.H.
        • Bigalke H.
        • Bergey G.K.
        • Neale E.A.
        • Hardegree M.C.
        • Nelson P.G.
        J. Neurochem. 1986; 47: 930-937
        • Erdal E.
        • Bartels F.
        • Binscheck T.
        • Erdmann G.
        • Frevert J.
        • Kistner A.
        • Weller U.
        • Wever J.
        • Bigalke H.
        Naunyn-Schmiedeberg's Arch. Pharmacol. 1995; 351: 67-78
        • Dolly J.O.
        • Lawrence G.W.
        • Foran P.
        Tranter H.S. Proceedings of Biomedical Aspects of Clostridial Neurotoxins, International Conference, Oxford. Center for Applied Microbial Research, Salisbury, UK1999: 97-102
        • Ashton A.C.
        • Dolly J.O.
        J. Neurochem. 2000; 74: 1979-1988