Mechanism of Action of OnabotulinumtoxinA in Chronic Migraine: A Narrative Review.

OBJECTIVE: To review the literature on the mechanism of action of onabotulinumtoxinA in chronic migraine. BACKGROUND: OnabotulinumtoxinA is a chronic migraine preventive treatment that significantly reduces headache frequency. The traditional mechanism described for onabotulinumtoxinA - reducing muscle contractions - is insufficient to explain its efficacy in migraine, which is primarily a sensory neurological disease. METHODS: A narrative literature review on the mechanism of action of onabotulinumtoxinA in chronic migraine. RESULTS: Following injection into tissues, onabotulinumtoxinA inhibits soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated vesicle trafficking by cleaving one of its essential proteins, soluble N-ethylmaleimide-sensitive fusion attachment protein (SNAP-25), which occurs in both motor and sensory nerves. OnabotulinumtoxinA inhibits regulated exocytosis of motor and sensory neurochemicals and proteins, as well as membrane insertion of peripheral receptors that convey pain from the periphery to the brain, because both processes are SNARE dependent. OnabotulinumtoxinA can decrease exocytosis of pro-inflammatory and excitatory neurotransmitters and neuropeptides such as substance P, calcitonin gene-related peptide, and glutamate from primary afferent fibers that transmit nociceptive pain and participate in the development of peripheral and central sensitization. OnabotulinumtoxinA also decreases the insertion of pain-sensitive ion channels such as transient receptor potential cation channel subfamily V member 1 (TRPV1) into the membranes of nociceptive neurons; this is likely enhanced in the sensitized neuron. For chronic migraine prevention, onabotulinumtoxinA is injected into 31-39 sites in 7 muscles of the head and neck. Sensory nerve endings of neurons whose cell bodies are located in trigeminal and cervical ganglia are distributed throughout the injected muscles, and are overactive in people with migraine. Through inhibition of these sensory nerve endings, onabotulinumtoxinA reduces the number of pain signals that reach the brain and consequently prevents activation and sensitization of central neurons postulated to be involved in migraine chronification. CONCLUSION: OnabotulinumtoxinA likely acts via sensory mechanisms to treat chronic migraine.

Lithium Improves Dopamine Neurotransmission and Increases Dopaminergic Protein Abundance in the Striatum after Traumatic Brain Injury.

Experimental models of traumatic brain injury (TBI) recapitulate secondary injury sequela and cognitive dysfunction reported in patients afflicted with a TBI. Impairments in neurotransmission are reported in multiple brain regions in the weeks following experimental TBI and may contribute to behavioral dysfunction. Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is an important mechanism for neurotransmitter exocytosis. We previously showed that lithium treatment attenuated hippocampal decreases in α-synuclein and VAMP2, enhanced SNARE complex formation, and improved cognitive performance after TBI. However, the effect of TBI on striatal SNARE complex formation is not known. We hypothesized lithium treatment would attenuate TBI-induced impairments in evoked dopamine release and increase the abundance of synaptic proteins associated with dopamine neurotransmission. The current study evaluated the effect of lithium (1 mmol/kg/day) administration on striatal evoked dopamine neurotransmission, SNARE complex formation, and proposed actions of lithium, including inhibition of GSK3ß, assessment of synaptic marker protein abundance, and synaptic proteins important for dopamine synthesis and transport following controlled cortical impact (CCI). Sprague-Dawley rats were subjected to CCI or sham injury and treated daily with lithium chloride or vehicle for 7 days post-injury. We provide novel evidence that CCI reduces SNARE protein and SNARE complex abundance in the striatum at 1 week post-injury. Lithium administration improved evoked dopamine release and increased the abundance of α-synuclein, D2 receptor, and phosphorylated tyrosine hydroxylase in striatal synaptosomes post-injury. These findings show that lithium treatment attenuated dopamine neurotransmission deficits and increased the abundance of synaptic proteins important for dopamine signaling after TBI.

Role of SNARE proteins in tumourigenesis and their potential as targets for novel anti-cancer therapeutics.

The function of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in cellular trafficking, membrane fusion and vesicle release in synaptic nerve terminals is well characterised. Recent studies suggest that SNAREs are also important in the control of tumourigenesis through the regulation of multiple signalling and transportation pathways. The majority of published studies investigated the effects of knockdown/knockout or overexpression of particular SNAREs on the normal function of cells as well as their dysfunction in tumourigenesis promotion. SNAREs are involved in the regulation of cancer cell invasion, chemo-resistance, the transportation of autocrine and paracrine factors, autophagy, apoptosis and the phosphorylation of kinases essential for cancer cell biogenesis. This evidence highlights SNAREs as potential targets for novel cancer therapy. This is the first review to summarise the expression and role of SNAREs in cancer biology at the cellular level, their interaction with non-SNARE proteins and modulation of cellular signalling cascades. Finally, a strategy is proposed for developing novel anti-cancer therapeutics using targeted delivery of a SNARE-inactivating protease into malignant cells.

General aspects and recent advances on bacterial protein toxins.

Bacterial pathogens produce protein toxins to influence host-pathogen interactions and tip the outcome of these encounters toward the benefit of the pathogen. Protein toxins modify host-specific targets through posttranslational modifications (PTMs) or noncovalent interactions that may inhibit or activate host cell physiology to benefit the pathogen. Recent advances have identified new PTMs and host targets for toxin action. Understanding the mechanisms of toxin action provides a basis to develop vaccines and therapies to combat bacterial pathogens and to develop new strategies to use toxin derivatives for the treatment of human disease.

Endocytic SNAREs are involved in optimal Coxiella burnetii vacuole development.

Coxiella burnetii is a Gram-negative intracellular bacterium. As previously described, both the endocytic and the autophagic pathways contribute to the maturation of Coxiella replicative vacuoles (CRVs). The large CRVs share the properties of both phagolysosomal and autophagolysosomal compartments. Vamp3, Vamp7 and Vamp8 are v-SNAREs involved in the endocytic pathway which participate mainly in the fusion between endosomes and lysosomes. In the present study we observed that Vamp7 interacts with C. burnetii at different infection times (1 h-48 h p.i.). We have determined that a truncated mutant of Vamp7 (Vamp7 NT) and a siRNA against this SNARE protein affects the optimal development of CRVs, suggesting that Vamp7 mediates fusion events that are required for the biogenesis of CRVs. Indeed, we have observed that overexpression of Vamp7 NT inhibited the heterotypic fusion with lysosomes and the homotypic fusion between individual Coxiella phagosomes and CRVs. Moreover, we have detected in the vacuole membrane, at different infection times, the Vamp7 partners (Vti1a and Vti1b). Interestingly, treatment with chloramphenicol reduced the colocalization between C. burnetii and Vamp7, Vti1a or Vti1b, indicating that the recruitment of these SNAREs proteins is a bacteria-driven process that favours the CRV biogenesis, likely by facilitating the interaction with the endolysosomal compartment.

[Botulinum neurotoxin--a therapy in migraine]. / Botulinum neurotoxin--a terápia migrénben.

Although migraine is a common, paroxysmal, highly disabling disorder, the primary cause and the pathomechanism of migraine attacks are enigmatic. Experimental results suggest that activation of the trigeminovascular system is crucial in its pathogenesis. This activation leads to the release of vasoactive neuropeptides (calcitonin gene-related peptide - CGRP, and substance P - SP) and to neurogenic inflammation, and peripheral and central sensitisation are expressed. Botulinum neurotoxin-A (BoNT-A), a potent toxin produced by Clostridium botulinum, affects the nervous system through specific cleavage of the soluble NSF-attachment protein receptor complex (SNARE), like synaptosomal-associated protein of 25 kDa (SNAP-25). The result of this multistage process is blockade of the presynaptic release of pain neurotransmitters such as CGRP, SP and glutamate. A pooled analysis of the data from two programmes of Phase 3 Research Evaluating Migraine Prophylaxis Therapy (PREEMPT 1 and 2) with BoNT-A in chronic migraine demonstrated significant benefit of BoNT-A over placebo with regard to the numbers of headache days and migraine episodes. BoNT-A diminished the frequency of acute headache pain medication intake, and resulted in reductions in headache impact and improvements in scores on the Migraine-Specific Quality of Life Questionnaire. The treatments with BoNT-A proved safe and were well tolerated.

Association of botulinum neurotoxins with synaptic vesicle protein complexes.

Botulinum neurotoxins (BoNTs) elicit flaccid paralysis by cleaving SNARE proteins within peripheral neurons. BoNTs are classified into seven serotypes, termed A-G, based on antibody cross-neutralization. Clostridia produce BoNTs as single-chain toxins that are cleaved into a di-chain protein that comprises an N-terminal zinc metalloprotease domain that is linked by a disulfide bond to the C-terminal translocation/receptor-binding domain. BoNT/A and BoNT/B utilize synaptic vesicle protein 2 (SV2) and synaptotagmin, respectively, as receptors for entry into neurons. Using affinity chromatography, BoNT/A and BoNT/B were found to bind a synaptic vesicle protein complex in CHAPS extracts of synaptic vesicles. Mass spectroscopy identified synaptic vesicle protein 2, synaptotagmin I, synaptophysin, vesicle-associated membrane protein 2, and the vacuolar ATPase-proton pump as components of the BoNT-synaptic vesicle protein complex. BoNT/A and BoNT/B possessed unique density-gradient profiles when bound to synaptic vesicle protein complexes. The identification of BoNT/A and BoNT/B bound to synaptic vesicle protein complexes provides insight into the interactions of BoNT and neuronal receptors.

Receptor and substrate interactions of clostridial neurotoxins.

The high potency of clostridial neurotoxins relies predominantly on their neurospecific binding and specific hydrolysis of SNARE proteins. Their multi-step mode of mechanism can be ascribed to their multi-domain three-dimensional structure. The C-terminal H(CC)-domain interacts subsequently with complex polysialo-gangliosides such as GT1b and a synaptic vesicle protein receptor via two neighbouring binding sites, resulting in highly specific uptake of the neurotoxins at synapses of cholinergic motoneurons. After its translocation the enzymatically active light chain specifically hydrolyses specific SNARE proteins, preventing SNARE complex assembly and thereby blocking exocytosis of neurotransmitter.

Translocation of botulinum neurotoxin light chain protease by the heavy chain protein-conducting channel.

Clostridial botulinum neurotoxins (BoNTs) inhibit synaptic exocytosis; intoxication requires the di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol, colocalizing it with the substrate SNARE proteins. We investigate the dynamics of protein translocation across membranes using a sensitive single-molecule assay to track translocation events with millisecond resolution on lipid bilayers and on membrane patches of Neuro 2A cells. Translocation of BoNT/A LC by the HC is observed in real time as changes of channel conductance: the channel is occluded by the light chain during transit, and open after completion of translocation and release of cargo, acting intriguingly similar to the protein-conducting/translocating channels of the endoplasmic reticulum, mitochondria, and chloroplasts. Our findings support the notion of an interdependent, tight interplay between the HC transmembrane chaperone and the LC cargo that prevents LC aggregation and dictates the productive passage of cargo through the channel and completion of translocation. The protein-conducting channel of BoNT, a key element in the process of neurotoxicity, emerges therefore as a target for antidote discovery - a novel paradigm of paramount significance to health science and biodefense.

Physiology of airway mucus secretion and pathophysiology of hypersecretion.

Mucus secretion is the first-line defense against the barrage of irritants that inhalation of approximately 500 L of air an hour brings into the lungs. The inhaled soot, dust, microbes, and gases can all damage the airway epithelium. Consequently, mucus secretion is extremely rapid, occurring in tens of milliseconds. In addition, mucus is held in cytoplasmic granules in a highly condensed state in which high concentrations of Ca(2+) nullify the repulsive forces of the highly polyanionic mucin molecules. Upon initiation of secretion and dilution of the Ca(2+), the repulsion forces of the mucin molecules cause many-hundred-fold swelling of the secreted mucus, to cover and protect the epithelium. Secretion is a highly regulated process, with coordination by several molecules, including soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) proteins, myristoylated alanine-rich C kinase substrate (MARCKS), and Munc proteins, to dock the mucin granules to the secretory cell membrane prior to exocytosis. Because mucus secretion appears to be such a fundamental airway homeostatic process, virtually all regulatory and inflammatory mediators and interventions that have been investigated increase secretion acutely. When given longer-term, many of these same mediators also increase mucin gene expression and mucin synthesis, and induce goblet cell hyperplasia. These responses induce (in contrast to the protective effects of acute secretion) long-term, chronic hypersecretion of airway mucus, which contributes to respiratory disease. In this case the homeostatic, protective function of airway mucus secretion is lost, and, instead, mucus hypersecretion contributes to pathophysiology of a number of severe respiratory conditions, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis.

Mechanism of arachidonic acid action on syntaxin-Munc18.

Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.

A second SNARE role for exocytic SNAP25 in endosome fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicle-plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.

Early, transient increase in complexin I and complexin II in the cerebral cortex following traumatic brain injury is attenuated by N-acetylcysteine.

Alteration of excitatory neurotransmission is a key feature of traumatic brain injury (TBI) in which extracellular glutamate levels rise. Although increased synaptic release of glutamate occurs at the injury site, the precise mechanism is unclear. Complexin I and complexin II constitute a family of cytosolic proteins involved in the regulation of neurotransmitter release, competing with the chaperone protein alpha-SNAP (soluble N-ethylmaleimide-sensitive factor-attachment protein) for binding to the synaptic vesicle protein synaptobrevin as well as the synaptic membrane proteins SNAP-25 and syntaxin, which together form the SNAP receptor (SNARE) complex. Complexin I is predominantly a marker of axosomatic (inhibitory) synapses, whereas complexin II mainly labels axodendritic and axospinous synapses, the majority of which are excitatory. In order to examine the role of these proteins in TBI, we have studied levels of both complexins in the injured hemisphere by immunoblotting over a time period ranging from 6 h to 7 days following lateral fluid-percussion brain injury in the rat. Transient increases in the levels of complexin I and complexin II proteins were detected in the injured cerebral cortex 6 h following TBI. This increase was followed by a decrease of complexin I in the injured cortex and hippocampus, and a decrease in both complexins in the injured thalamus region at day 3 and day 7 post-injury. The early, transient increase in the injured cortex was completely blocked by N-acetylcysteine (NAC) administered 5 min following trauma, suggesting an involvement of oxidative stress. Neuronal loss was also reduced in the injured hemisphere with post-TBI NAC treatment. Our findings suggest a dysregulation of both inhibitory and excitatory neurotransmission following traumatic injury that is responsive to antioxidant treatment. These alterations in complexin levels may also play an important role in neuronal cell loss following TBI, and thus contribute to the pathophysiology of cerebral damage following brain injury.

Internalization and mechanism of action of clostridial toxins in neurons.

Botulinum toxins are metalloproteases that act inside nerve terminals and block neurotransmitter release via their activity directed specifically on SNARE proteins. This review summarizes data on botulinum toxin modes of binding, sites of action, and biochemical activities. Their use in cell biology and neuroscience is considered, as well as their therapeutic utilization in human diseases characterized by hyperfunction of cholinergic terminals.