Structural and Functional Interactions between Transient Receptor Potential Vanilloid Subfamily 1 and Botulinum Neurotoxin Serotype A.

BACKGROUND: Botulinum neurotoxins are produced by Clostridium botulinum bacteria. There are eight serologically distinct botulinum neurotoxin isoforms (serotypes A-H). Currently, botulinum neurotoxin serotype A (BoNT/A) is commonly used for the treatment of many disorders, such as hyperactive musculoskeletal disorders, dystonia, and pain. However, the effectiveness of BoNT/A for pain alleviation and the mechanisms that mediate the analgesic effects of BoNT/A remain unclear. To define the antinociceptive mechanisms by which BoNT/A functions, the interactions between BoNT/A and the transient receptor potential vanilloid subfamily 1 (TRPV1) were investigated using immunofluorescence, co-immunoprecipitation, and western blot analysis in primary mouse embryonic dorsal root ganglion neuronal cultures. RESULTS: 1) Three-week-old cultured dorsal root ganglion neurons highly expressed transient TRPV1, synaptic vesicle 2A (SV2A) and synaptosomal-associated protein 25 (SNAP-25). SV2A and SNAP-25 are the binding receptor and target protein, respectively, of BoNT/A. 2) TRPV1 colocalized with both BoNT/A and cleaved SNAP-25 when BoNT/A was added to dorsal root ganglia neuronal cultures. 3) After 24 hours of BoNT/A treatment (1 nmol/l), both TRPV1 and BoNT/A positive bands were detected in western blots of immunoprecipitated pellets. 4) Blocking TRPV1 with a specific antibody decreased the cleavage of SNAP-25 by BoNT/A. CONCLUSION: BoNT/A interacts with TRPV1 both structurally and functionally in cultured mouse embryonic dorsal root ganglion neurons. These results suggest that an alternative mechanism is used by BoNT/A to mediate pain relief.

Neuritogenic actions of botulinum neurotoxin A on cultured motor neurons.

Botulinum neurotoxins (BoNTs) are extremely potent neuromuscular poisons that act through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein cleavage to inhibit neurotransmitter release. The ability of BoNT serotype A (BoNT/A) to eliminate localized transmitter release at extremely low doses is well characterized. In the current study, we investigated the less understood characteristic of BoNT/A to induce nerve outgrowth, sometimes referred to as sprouting. This phenomenon is generally considered a secondary response to the paralytic actions of BoNT/A, and other potential factors that may initiate this sprouting have not been investigated. Alternatively, we hypothesized that BoNT/A induces sprouting through presynaptic receptor activation that is independent of its known intracellular actions on the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) synaptosomal associated protein of 25 kDa (SNAP-25). To test this, the effects of BoNT/A application on neurite outgrowth were examined using primary cultures enriched with motor neurons isolated from embryonic mouse spinal cord. In this system, BoNT/A potently stimulated neuritogenesis at concentrations as low as 0.01 nM. The neuritogenic effects of BoNT/A exposure were concentration dependent and antagonized by Triticum vulgaris lectin, a known competitive antagonist of BoNT. Similar results were observed with the isolated BoNT/A binding domain, revealing that neuritogenesis could be initiated solely by the binding actions of BoNT/A. In addition, the presence or absence of SNAP-25 cleavage by BoNT/A was not a determinant factor in BoNT/A-induced neuritogenesis. Collectively, these results suggest that binding of BoNT/A to the motor neuronal membrane activates neuritogenesis through as yet undetermined intracellular pathway(s), independent of its known action on vesicular release.

Molecular targets of botulinum toxin at the mammalian neuromuscular junction.

The molecular targets of botulinum neurotoxins (BoNTs) are SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins necessary for neurotransmitter release. BoNT are powerful therapeutic agents in the treatment of numerous neurological disorders. The goals of this study were to (1) assess toxin diffusion by measuring substrate cleavage in adjacent and distant muscles, and (2) characterize the clinical course using SNARE protein chemistry. A small volume of BoNT/A was injected unilaterally into the mouse gastrocnemius muscle. Motor impairment was limited to the toxin-treated limb. No systemic illness or deaths occurred. At five time points, a subset of mice were killed, and muscles from both hindlimbs, and the diaphragm, were collected. Protein samples were examined for changes in SNAP-25 (synaptosomal-associated protein of Mr = 25 kDa) using immunochemistry. SNAP-25 cleavage product was noted in the toxin-treated limb as early as 1 day postinjection and continued through day 28. Onset and peak levels of substrate cleavage corresponded to the onset and peak clinical response. Cleavage was observed in adjacent and distant muscles, demonstrating that substrate cleavage is a sensitive indicator of toxin diffusion. Significant increases in full-length SNAP-25 and vesicle-associated membrane protein II were evident early in the impaired limb and continued through day 28. The increased SNARE protein most likely originates from nerve terminal sprouts.

Expression of P2X6, a purinergic receptor subunit, is affected by dietary zinc deficiency in rat hippocampus.

The purpose of these experiments was to determine whether dietary zinc depletion affected protein expression in the hippocampus. Eleven weanling Sprague-Dawley male rats (21 d) were fed the AIN-93G diet containing 1.5 ppm zinc and supplemented with 30 ppm of zinc in the drinking water. After 1 wk, the rats were randomly divided into three groups: control (n=3), pair fed (n=3), and zinc restricted (n=5). All groups consumed the same diet. The zinc-restricted group consumed water containing no zinc. The rats were sacrificed 3 wk later. Chelatable zinc levels in the hippocampus, as measured by N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) staining, were significantly reduced in the zinc-restricted group. Analysis of hippocampal protein expression by two-dimensional electrophoresis (2DE) revealed increased expression of the P2X6 purinergic receptor in the zinc-restricted rats, as determined by MALDI mass spectrometry (MS) and database analysis. The data provided evidence for the dual effects of dietary zinc deficiency on the hippocampus, reducing ionic zinc levels and stimulating protein expression. The role the P2X6 receptor plays in the physiological response of the hippocampus to zinc depletion remains to be determined.

Botulinum neurotoxin: the neuromuscular junction revisited.

Botulinum neurotoxin is the neuromuscular poison that is responsible for the fatal disease botulism. This toxin is also a valued therapeutic agent in the treatment of an increasing number of neuromuscular disorders. Unfortunately, in the wrong hands, botulinum neurotoxin is also a deadly biological "weapon. The diverse health consequences of botulinum neurotoxin combined with the increased threat of bioterrorism underscore the profound importance of understanding exactly how this toxin exerts its effects on the clinically relevant mammalian target site, the neuromuscular junction. Despite the fact that a great deal has been learned about the cellular actions of botulinum neurotoxin during the past three decades, questions still remain. For example, what protein or proteins mediate transport of the toxin into the cholinergic nerve terminal? What factors control the duration of toxin action in the nerve terminal? Until recently, scholarly pursuit of such questions was technically challenging in neuromuscular tissues. Recent advancements in biotechnology have now made it feasible to pursue these important issues at the neuromuscular junction and to correlate biochemical studies in nontarget tissues with clinically relevant functional outcomes. This narrative reviews our current understanding of the actions of botulinum neurotoxin at the neuromuscular junction, presents recent findings from our own work in neuromuscular tissues, and encourages future studies regarding botulinum neurotoxin at its target site.