The Janus faces of botulinum neurotoxin: sensational medicine and deadly biological weapon.

The botulinum neurotoxins are the most dangerous toxins known (BoNTs serotypes A-G) and induce profound flaccid neuromuscular paralysis by blocking nerve-muscle communication. Poisoned motoneurons react by emitting a sprouting network known to establish novel functional synapses with the abutting muscle fiber. Understanding how our motoneurons are capable of bypassing such transmission blockade, thereby overcoming paralysis, by an astonishing display of plasticity is one of the research goals that have numerous therapeutic ramifications. This Mini-Review aims at giving a brief update on the recent discoveries regarding the molecular mechanism of botulinum toxins intoxication. Curing botulism still is a challenge once the toxin has found his way inside motoneurons. In view of the potential use of botulinum toxins as biological weapon, more research is needed to find efficient ways of curing this disease.

A novel polyclonal antibody specific for the Na(v)1.5 voltage-gated Na(+) channel 'neonatal' splice form.

Voltage-gated Na(+) channel (VGSC) diversity is achieved through a number of mechanisms: multiple subunits, multiple genes encoding the pore-forming VGSC alpha-subunit and multiple gene isoforms generated by alternative splicing. A major type of VGSCalpha alternative splicing is in D1:S3, which has been proposed to be developmentally regulated. We recently reported a D1:S3 spliced form of Na(v)1.5 in human metastatic breast cancer cells. This novel 'neonatal' isoform differs from the counterpart 'adult' form at seven amino acids (in the extracellular loop between S3-S4 of D1). Here, we generated an anti-peptide polyclonal antibody, named NESOpAb, which specifically recognised 'neonatal' but not 'adult' Na(v)1.5 when tested on cells specifically over-expressing one or other of these Na(v)1.5 spliced forms. The antibody was used to investigate developmental expression of 'neonatal' Na(v)1.5 (nNa(v)1.5) in a range of mouse tissues by immunohistochemistry. Overall, the results were consistent with nNa(v)1.5 protein being more abundantly expressed in selected tissues (particularly heart and brain) from neonate as compared to adult animals. Importantly, NESOpAb blocked functional nNa(v)1.5 ion conductance when applied extracellularly at concentrations as low as 0.05 ng/ml. Possible biological and clinical applications of NESOpAb are discussed.

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.

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, approximately 10, approximately 2, and approximately 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.

Insights into a basis for incomplete inhibition by botulinum toxin A of Ca2+-evoked exocytosis from permeabilised chromaffin cells.

Inhibition of regulated exocytosis by botulinum toxins type A and B was studied in chromaffin cells. Both virtually abolished catecholamine release triggered from intact cells by depolarising stimuli, whereas the blockade by type A, but not B, was only partial after cell permeabilisation and direct stimulation of exocytosis by Ca(2+). Botulinum toxin A did not alter the [Ca(2+)]-dependency of exocytosis in permeabilised cells but, rather, proportionally reduced the amount of release at each concentration tested. Likewise, this toxin decreased the extents of Ca(2+)-induced structural changes in SNAP-25, synaptobrevin and syntaxin (known collectively as SNAREs), whilst leaving their [Ca(2+)]-sensitivity unaltered. Thus, botulinum toxin A does not reduce the Ca(2+)-sensitivity of the exocytosis sensor, but hinders transmission of the signal to the SNAREs which mediate fusion.