Facoltà di Scienze Matematiche Fisiche e Naturali
Corso di Laurea Magistrale in
Biologia Applicata alla Biomedicina
(
curriculum Neurobiologico
)
Direct central action of botulinum neurotoxin type A at
cholinergic synapses following intramuscular injection
Relatore Candidato
Prof. Matteo Caleo Matteo Spinelli
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A mia madre, unico sostegno oltre ogni cosa.
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“I AM A SLOW WALKER, BUT I NEVER WALK BACK”
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Index
ABSTRACT ... 5
1 INTRODUCTION ... 7
1.1 CLOSTRIDIAL NEUROTOXINS ... 7
1.1.1 Structure of Botulinum Neurotoxins ... 8
1.2 MECHANISM OF ACTION OF BoNTs ... 11
1.2.1 Binding and specificity ... 12
1.2.2 Internalization into nerve terminal ... 14
1.2.3 Membrane translocation and release into the cytosol ... 14
1.2.4 Interchain disulfide reduction ... 16
1.2.5 SNARE protein cleavage ... 16
1.3 NEUROEXOCYTOSIS AND SNARE PROTEINS ... 17
1.4 MOLECOLAR TARGETS ... 20
1.5 DURATION OF ACTION ... 24
1.6 CLINICAL USES OF BoNTs ... 26
1.6.1 Dystonias ... 26
1.6.2 Spasticity ... 28
1.6.3 Pain ... 29
1.7 MEASURING BoNT/A CENTRAL EFFECT IN HUMANS ... 29
1.8 BoNT/A TRANSPORT: EXSPERIMENT IN THE RAT VISUAL SYSTEM ... 31
1.8.1 Anterograde propagation ... 31
1.8.2 Retrograde propagation ... 34
2 AIM OF THE THESIS ... 35
3 MATERIALS AND METHODS ... 36
3.1 ANIMALS ... 36 3.2 WHISKER-PAD INJECTIONS ... 36 3.3 AXOTOMY ... 37 3.4 ANTITOXIN INJECTIONS ... 37 3.5 HISTOLOGY ... 38 3.5.1 Fixative solution ... 38 3.5.2 Fixative procedure ... 38 3.6 IMMUNOHISTOCHEMISTRY ... 39
3.6.1 BoNT/A-cleaved SNAP-25 (SNAP/A) detection ... 40
3.6.2 Detection of the other synaptic markers ... 40
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3.7.1 Immunohistochemical quantification ... 40
3.7.2 Colocalization analysis ... 41
4 RESULTS ... 42
4.1 RETROGRADE PROPAGATION OF CLEAVED SNAP-25 TO THE FACIAL NUCLEUS: DIFFERENT KINETICS IN MOUSE AND RAT ... 42
4.2 EVIDENCE OF THE TRANSPORT OF THE CATALYTICALLY ACTIVE FORM OF BoNT/A ... 43
4.3 EVIDENCE FOR BoNT/A TRANSCYTOSIS ... 46
4.4 PREFERENTIAL ACTIVITY OF BoNT/A IN CHOLINERGIC TERMINALS ... 48
5 DISCUSSION ... 52
5.1 DETECTION OF BoNT/A EFFECT IN THE FACIAL NUCLEUS AND RETROGRADE TRANSPORT ... 52
5.2 RETROGRADE TRASPORT OF ACTIVE BoNT/A IN THE FACIAL NUCLEUS ... 53
5.3 TRANSCYTOSIS ... 54
5.4 PREFERENCE OF INTERNALIZATION ... 56
5.5 POSSIBLE CLINICAL IMPLICATIONS AND FUTURE PERSPECTIVES ... 58
5
ABSTRACT
Clostridial toxins are the most potent neurotoxins known. They include tetanus toxin (TeNT), that causes tetanus, and botulinum toxins (BoNT/s) that cause botulism. These two pathological conditions are characterized respectively by a spastic paralysis due to blockage of spinal cord inhibitory circuits, and a flaccid paralysis due to interference with acetylcholine release at the level of the neuromuscular junction.
Following endocytosis into the neuron and subsequent translocation in the cytosol, clostridial toxins exert their metalloproteasic activity against specific SNARE complex components, which are fundamental for the docking and fusion of synaptic vesicles. The cleavage of SNARE proteins indeed causes an arrest of neurotransmitter release. Botulinum toxin type A (BoNT/A) exerts a prolonged yet transient blockade of transmitter release at the neuromuscular junction. For this reason, it is widely used not only in cosmetics (for the removal of facial wrinkles) but also in the clinical treatment of pathologies characterized by hyperfunctionality of cholinergic terminals, such as spasticity and dystonia, and for treatment of chronic pain. The persistent clinical benefits of BoNT/A treatment even after the time window of neuromuscular blockade suggested the involvement of the central nervous system. Such central effects may be due to direct or indirect action of the toxin. Previous experimental data have demonstrated that BoNT/A can be trafficked along the axon of the motoneuron through a retrograde transport process, suggesting a direct central action of the toxin. In my thesis, I investigated whether the toxin was able to undergo transcytosis, i.e. entry into second-order neurons afferent to the motoneurons after transport, and the potential selectivity of such process.
After injecting BoNT/A into the whisker-pad of rats and mice, using an immunohistochemical assay, I detected significant levels of cleaved SNAP-25 (SNAP/A) in the nucleus facialis of the brainstem, i.e. the area containing the motoneurons projecting to the whiskers. I observed a different kinetics in the retrograde propagation of toxin effects between rat and mouse. Specifically, retrograde spread was faster in mice, with SNAP/A being detectable already at one day after injection. At 15 days after the injection, levels of SNAP/A were roughly comparable between the two species.
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To demonstrate whether the active toxin is transported (rather than the cleaved SNAP-25), I sectioned the facial nerve to block axonal transport. Accumulation of SNAP/A in the nucleus facialis was observed despite the axotomy, indicating transport of the the catalytically active BoNT/A.
To obtain compelling evidence for BoNT/A transcytosis, I performed intracerebroventricular injections of an antitoxin following peripheral injection of BoNT/A. The data showed a marked decrease in SNAP/A in animals treated with antitoxin compared to controls. Thus, the antitoxin was capable of specifically intercepting BoNT/A, demonstrating that the toxin physically leaves motoneurons to enter second-order neurons in the brainstem.
Finally, by double-label immunohistochemistry and signal colocalization analysis, I investigated the potential selectivity of transcytosis. I compared the presence of SNAP/A in cholinergic, GABAergic and glutamatergic synaptic inputs to motoneurons. I found a preferential localization of SNAP/A in the cholinergic terminals, accompanied by an increase in the size of the same terminals due to the accumulation of synaptic vesicles unable to fuse with the presynaptic membrane.
Altogether, these data these findings highlights cell-specific, direct central actions of BoNT/A which are important to fully understand its mechanisms of action and therapeutic effectiveness.
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1 INTRODUCTION
1.1 CLOSTRIDIAL NEUROTOXINS
Clostridium is a genus of sporulating and anaerobic Gram-positive, rod-shaped bacteria
that includes more than 150 species. These bacteria are widely distributed in the environment and in anaerobic regions of the intestines of several animals, where they are typically found as spores, which are resistant to physical and chemical stresses and can persist for long periods, until favourable conditions enable germination (Popoff et al., 2013; Johnson and Montecucco, 2008). Several clostridia, including Clostridium
difficile, Clostridium perfringens and Clostridium sordelli, are pathogenic, owing to the
release of protein toxins, but only a few species are neurotoxigenic. For example,
Clostridium tetani produces tetanus neurotoxin (TeNT), which blocks neurotransmitter
release in spinal cord interneurons and causes the spastic paralysis of tetanus (Schiavo, Matteoli and Montecucco, 2000). Clostridium tetani is strictly anaerobic because it does not possess the redox enzymes necessary to reduce oxygen. It is widespread in nature in the form of spores, which germinate under appropriate conditions of very low oxygen tension, slight acidity and availability of nutrients. Such conditions may be present in anaerobic wounds or skin ruptures or abrasions, where spores can germinate and produce a protein toxin that fills the bacterial cytosol and is released by autolysis.
The strains Clostridium botulinum, Clostridium butyrricum, Clostridium barati, and Clostridium argentinensis cause the flaccid paralysis and autonomic dysfunctions that
are typical of botulism, by intoxication of one of the eight neurotoxins (BoNTs) produced under anaerobic conditions. The family of Clostridial neurotoxins (CNT) comprises indeed eight antigenically distinct botulinum neurotoxins (BoNT/A–H) and tetanus neurotoxin. For six of the eight BoNT serotypes, several subtypes have been described based on differences in amino acid sequences. For BoNT/A, five subtypes (named A1 to A5) are currently known with different enzymatic activity and toxicological properties (Akaike et al., 2013; Whitemarsh et al., 2013).
Each clostridial neurotoxin is synthesized as a single polypeptide chain (150 kDa) that is subsequently cleaved by proteases, to yield a 50-kDa light (L) chain and 100-kDa heavy (H) chain linked via a single disulphide bond.
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1.1.1 Structure of Botulinum Neurotoxins
The clinical outcomes of TeNT and BoNTs are very different, almost opposite in their manifestations, but the effect of CNT at the nerve terminals is the result of a closely related protein structure. CNT are synthesized in the bacterial cytosol and released in the culture medium only after bacterial lysis. No protein is associated with TeNT, whereas BoNT are released in the form of multimeric complexes, with a set of nontoxic proteins coded by genes adjacent to the neurotoxin gene: these complexes are termed progenitor toxins (Inoue et al., 1996; Minton, 1995) (Fig. 1a-b).
Some BoNT-associated proteins have hemagglutinating activity (HA), in addition, a large non-toxic-non-hemagglutinating protein of 139 kDa (NTNH), coded for by a gene upstream to the BoNT gene, is always present. The NTNH produced by the various neurotoxigenic strains of Clostridia are more conserved than the corresponding BoNT themselves. Moreover, a remarkable feature uncovered by gene sequencing is that the 100-amino acid-long NH2-terminal region of NTNH is homologous to the corresponding region of BoNT (Minton, 1995). The NTNH protein is significantly similar to BoNT/A and BoNT/B sequences (~20%), but lacks the HExxH zinc binding motif characteristic of Clostridial neurotoxin metalloproteases (Rawlings and Barrett, 1991; Schiavo et al., 1992b,c). The NH2-terminal region of the two proteins are independent domains with a strong tendency to dimerized (Schiavo, 2000). Such a structure suggests that NTNH shields and protects the BoNTs molecule from proteolytic and other chemical attacks (Miyata et al., 2009; Gu et al., 2012). This is particularly significant considering that this heterodimer is released within decaying biologic materials and it has to pass through the gastrointestinal tract that is rich of proteases and which is the most common portal of entry of the BoNTs into the animal body. Botulinum neurotoxin in the form of progenitor toxins is more stable than isolated BoNT to proteolysis and denaturation induced by temperature, solvent removal, or acid pH (Chen et al., 1998; Sakagushi, 1983). Progenitor toxins that survive the harsh conditions of the stomach reach the intestine, where the slightly alkaline pH induces their dissociation and releases the BoNT, which is then transcytosed to the mucosal side of the intestinal epithelium (Maksymowych and Simpson, 1998).
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The crystallographic structure of BoNT/A has been recently determined (Fig. 1a). The toxin structure reveals three distinct functional domains, a unique hybrid of previously characterized structural motifs and new insight into this protein's mechanism of toxicity (Fig. 2). There is complete agreement with the three-domain structural model of CNT.
Figure 1 Structure isolated BoNT molecules and BoNT complex. A) Crystal structure of botulinum neurotoxin A1 (BoNT/A1), showing its associated electrical dipole and the organization of individual toxin domain. B) Crystal structure of BoNT/A1 in complex with the NTNHA/A1 protein. (Rossetto et al., 2014)
10 Figure 2 Three-domain structure of clostridial neurotoxins.
Despite their amino acid sequence variability and immunologic differences, all BoNT serotypes display a similar molecular architecture. They are produced as inactive, single chain polypeptides of 150 KDa, which are nicked by proteases within a loop subtended by a strictly conserved disulfide bond. This cleavage originates the mature (and pharmacologically active) toxin. BoNT/A consist of three 50 kDa domains: an NH2-terminal domain endowed with zinc-endopeptidase activity; a membrane translocation domain characterized by the presence of two 10-nm-long a-helices, which are reminiscent of similar elements present in colicin and in the influenza virus hemagglutinin. In addition, a binding domain composed of two unique subdomains similar to the legume lectins and Kunitz inhibitor (Lacy at al., 1998).
The NH2-terminal domain is called L chain (light chain, 50 kDa) and it is a
metalloprotease with its active site buried within the core of the structure. Precisely a dependent Zn2+ endopeptidase (MEROPS Data Base: M27.002) that is inactive when
disulfide bonded to the rest of the molecule; its activity is expressed after reduction of the interchain disulfide bond. The active site zinc atom (motif HExxH) (Schiavo et al., 1992b,c) is coordinated by two histidine residues, a water molecule bound to a conserved glutamate residue and by the carboxylate group of another glutamate, with the likely participation of a conserved tyrosine (residue numbering corresponds to TeNT). The heavy chain (H 100 kDa) connected by disulfide bond, consists of two 50-kDa domains: the aminoterminal part, Hn (central domain) and the carboxy-terminal
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The Hn is responsible for the membrane translocation of the L chain into the neuronal
cytosol (Montal, 2010). The membrane translocation domain of BoNT/A has a cylindrical shape determined by the presence of a pair of unusually long and twisted 10-nm-long a-helices, reminiscent of the a-helices hairpin of colicin (Wiener et al., 1997). The COOH- terminal part (Hc) is mainly responsible of the interaction of BoNTs
with unmyelinated areas of motoneurons, ensuring a rapid and strong interaction of the toxin with peripheral cholinergic nerve endings (Dolly et al., 1984; Binz and Rummel, 2009; Rossetto et al., 2014).
The Hc domain consist of two distinct subdomains: the NH2-terminal half (Hc-N, 25
kDa) and the C-terminal half (Hc-C, 25 kDa), with little protein-protein contacts among
them. Hc-C is responsible for the neurospecific binding to a polysialoganglioside and to
the luminal domain of a synaptic vesicle protein (Binz and Rummel, 2009; Rummel, 2013). Such binding leads to the ensuing internalization and trafficking of the toxin within endocytic compartments. This is initiated by the retrieval of synaptic vesicles after the release of their contened neurotransmitter (Binz and Rummel, 2009; Rummel, 2013). The Hc-N folds similarly to sugar binding proteins of the lectin family,
but its specific function is not known, although there is evidence indicating that it may improve BoNTs adhesion to the presynaptic membrane by interacting with anionic lipids (Muraro et al., 2009; Montal, 2010; Zhang and Varnum, 2012; Zhang et al., 2013). The Hc-C contains a modified β-trefoil folding motif present in several proteins involved
in recognition and binding functions such as interleukin-1, fibroblast growth factor, and Kunitz-type trypsin inhibitors. Its sequence is poorly conserved among CNT. Removal of Hc-N from HC does not reduce HC nerve membrane binding, whereas
deletion of only 10 residues from the COOH terminus abolishes its binding to spinal cord neurons (Halpern and Loftus, 1993).
1.2 MECHANISM OF ACTION OF BoNTs
The structural organization of CNT is functionally related to the mechanism of neuronal intoxication. The mechanism of nerve terminal intoxication by the BoNTs is divided into five major steps (Fig. 3 summarizes such main steps): 1) binding to nerve terminals, 2) internalization within an endocytic compartment, 3) low pH-driven translocation of the L chain across the vesicle membrane, 4) release of the L chain in
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the cytosol by reduction of the inter-chain disulfide bond, and 5) cleavage of SNAREs with ensuing blockade of neurotransmitter release and neuroparalysis.
Figure 3 A multi-step botulinum neurotoxins intoxication process. The L chain (red) is released from the HN domain by the action of the thioredoxin reductasethioredoxin system (TrxR-Trx, blue and dark blue) and Hsp90 (mud colour), which reduce the interchain disulfide bond (orange) and avoid the aggregation of the protease.(Pirazzini et al., 2017).
1.2.1 Binding and specificity
The first step is represented from the neuro-specific binding. From the site of production or absorption BoNTs diffuse in body fluid as lymphatic or blood circulation, reaching and binding to the presynaptic plasma membrane of skeletal and autonomic cholinergic nerve terminals, that represents an infinitesimal part of the total surface area of cells and tissues exposed to body fluids. However, the toxin is not able to cross the blood brain barrier (Simpson 2013). Such neurospecificity and affinity of binding, together with their catalytic activity, is at the basis of the BoNTs toxicity and, at the same time, of their pharmacological and therapeutic use. BoNTs interact with two independent receptors of the presynaptic membrane: a polysialoganglioside (PSG) and the glycosylated luminal domain of a synaptic vesicle protein that mediates the following step of internalization (Montecucco, 1986). PSG is the first presynaptic receptor that BoNT contacts on the nerve terminal (Van Heyningen, 1959; Simpson and Rapport, 1971) and the PSG molecules are present at a high density on the presynaptic membrane.
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They are organized in microdomains that also include glycoproteins, and their oligosaccharide portion (which is the BoNT-binding moiety). PSGs also influence transmembrane signalling, endocytosis and vesicle trafficking (Simpson k. and Toomre, 2000; Prinetti et al., 2009). Thus, PSGs are perfectly equipped to function as “antennae” that capture BoNTs. Together with sphingomyelin, cholesterol, and some proteins, PSG form anionic microdomains in the plasma membrane (Simons and Toomre, 2000; Sonnino et al., 2007; Prinetti et al., 2009). Accordingly, BoNTs bind complex PSGs whose large oligosaccharide head group projects at a distance from the membrane surface and it is flexible and negatively charged. There is evidence that anionic microdomains of the presynaptic membrane, including PSG, may orient the electric dipole associated to the BoNT molecules whose positive end is significantly located around the PSG binding site. The binding is probably rapid, as it is likely to be controlled only by the rate of diffusion. The observation that neuronal stimulation enhances the toxicity of BoNTs (Hughes and Whaler, 1962; Keller et al., 2004) and that BoNT/B binds synaptotagmin in vitro (Nishiki et al., 1994), led to suggestion that BoNTs endocytosis is facilitated by a protein receptor consisting of luminal domain of a synaptic vesicle (SV) protein (Montecucco and Schiavo, 1995). This second binding provides the high affinity necessary to bind very low amounts of BoNT (during botulism 10-13/10-14 M). Making the vesicle interior available for the binding, also contributes to
account for the efficacy of BoNT in treating human syndromes characterized by hyperactive nerve terminals. This because the NMJs of these muscles have a higher rate of SVs exoendocytosis, which favours toxin uptake.
Following attachment to PSG, BoNT/B1, BoNT/DC and BoNT/G bind to segment of synaptotagmin (Syts) I and II, two proteins integral to the synaptic vesicle membrane (Dong et al., 2007; Rummel et al., 2007). The Syt binding domain is exposed at the synaptic terminal during exocytosis. BoNT/A1 and BoNT/E1 bind specifically two different segments of the fourth luminal loop of SV2, a multispanning integral protein of synaptic vesicles (Dong et al., 2006; Mahrhold et al., 2006; Binz and Rummel, 2009; Benoit et al., 2014). Three isoforms of SV2 (A,B,C) are expressed at motor nerve terminals, but SV2C appears to be the one binding BoNT/A1 more strongly in vitro. Moreover, glycosylated residues are comprised within the toxin binding area of SV2 (Benoit et al., 2014) and are clinically relevant. In fact, a different pattern of
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glycosylation among individuals would provide a simple explanation for the variable sensitivity of different patients to BoNT/A1 injection, which is often observed in clinical settings. Clearly, this variability might also be applicable to different vertebrate species.
1.2.2 Internalization into nerve terminal
As shown in figure 3, the second step of nerve terminal intoxication involves BoNT internalization. The dual binding interaction with PSG and synaptic vesicle receptors (Syt or SV2, depending on the toxin serotype) increases the strength of BoNT interactions with the membrane, which is the product of the two binding affinities (Montecucco, 1986). In both cultured neurons and in vivo, BoNT/A1 rapidly enters the synaptic vesicle lumen and the number of toxin molecules per vesicle probably either one or two (Colasante et al., 2013; Harper et al., 2011), correlates with the number of SV2 molecules in the synaptic vesicle membrane (Takamori et al., 2006). The rate of entry for BoNT/A1 correlates with the rate of synaptic vesicle endocytosis and with the rate of paralysis of the mouse phrenic nerve hemidiaphragm, which is the standard NMJ used to test the potency of BoNTs (Saheki et al., 2012; Wohlfarth et al., 1997; Rasetti-Escargueil et al., 2011)
The mechanism of internalization of other BoNTs remains to be established, but their ability to rapidly paralyse the mouse phrenic nerve hemidiaphragm suggests that they all use the synaptic vesicle as a “Trojan horse” to enter motor neuron terminals in vivo. By contrast, a marginal amount of toxin was also found in early endosome and multivesicular bodies within hippocampal cultures (Harper et al., 2011), suggesting that in cultured CNS neurons other trafficking routes might contribute to toxin entry particularly at the very high concentrations that are frequently used in the laboratory.
1.2.3 Membrane translocation and release into the cytosol
BoNTs translocate their L chain into the cytosol from an acidic intracellular compartment, and the process can be blocked by different amines and by bafilomycin A1, a specific inhibitor of the v-ATPase (Simpson, 1983; Williamson and Neale, 1994; Keller et al., 2004). However, the lumen of the SV inside the nerve terminal is not experimentally accessible, making the study of this process difficult. Studies carried out
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in the past decade have provided considerable insight and have led to the proposal of a molecular model for this process (Montal, 2010; Fischer, 2013; Rossetto et al., 2014). It was found that the translocation of L chain takes place between pH 4.5 and 6 (Pirazzini et al., 2011) and that the entire translocation process is rapid (few minutes at 37°C) and strongly temperature dependent (Pirazzini et al., 2013c). BoNT initially binds to its two receptors (PSG and SV2 or Syt) inside the synaptic vesicle lumen, which has a neutral pH, immediately after endocytosis. An essential component of SV is the electrogenic v-ATPase, which injects protons into the lumen, generating a transmembrane pH gradient of 1.4 pH units and an electrical gradient of +39 mV (Parsons, 2000). By using fluorescent synaptophluorins, the luminal pH of SV was estimated to be 5.8 pH units (Miesenbock et al., 1998). The vesicular ATPase thus pumps protons into the synaptic vesicle and the luminal pH becomes progressively more acidic. As the pH lowers, some conserved high pKa carboxylates of BoNT become protonated on the face of the molecule containing the interchain disulfide bond, which acquires a net positive charge, as indicated by bioinformatics and mutagenesis analysis. Remarkably, the opposite face is devoid of conserved high pK protonatable residues (Pirazzini et al., 2011, 2013b). Consequently, the BoNT molecule, with its positively charged surface, falls onto the anionic surface of the membrane generating a lipid-protein complex. The protonation of carboxylate residues of lower pKa values triggers a process of structural change involving the L and H chains together with membrane lipids, leading to the formation of an ion channel. There is a consensus that the H chain acts as a sort of transmembrane chaperone for the translocation of the L chain across the membrane (Koriazova and Montal, 2003; Montal, 2010; Fischer, 2013). Different molecular roles of the H chain can be envisaged, and two possibilities with a range of intermediate cases are mentioned here: 1) that of a protein conducting channel that translocate the unfolded L chain (Collier, 2009). 2) That the toxin forms a “molten globule”, a protein state that is known to be capable of interacting with the hydrophobic membrane interior (Ptitsyn et al., 1990; van der Goot et al., 1991). Such molten globule would insert into the membrane together with anionic lipids and would deliver the L chain to the other side of the membrane, whereas the H chain would assemble an ion conducting channel. In any case, the belt has to be unfastened to
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permit the passage of the L chain on the cytosolic face of the membrane (Fischer and Montal, 2007).
1.2.4 Interchain disulfide reduction
The importance of the interchain disulfide for the toxicity of clostridial neurotoxins is demonstrated by the fact that reduced toxins are inactive (Schiavo et al., 1990; de Paiva et al., 1993; Fischer and Montal, 2007). At the cellular level, Fischer and Montal (2007) demonstrated that the premature reduction of this S-S bond, at any stage before its exposure to the cytosol, aborts the L chain translocation. Therefore, this domain must emerge on the cytosolic side before reduction takes place. The reduction of protein disulphide bonds is catalysed in the cell cytosol by different enzymatic systems, including glutaredoxins, thioredoxins and other systems (Mayer et al., 2009; Berndt et al., 2008). The NADPH-thioredoxin reductase (TrxR)-thioredoxin (Trx) system is a major redox system of the cell that reduces protein disulphides. TrxR and Trx were shown to be extrinsic proteins of the cytosolic side of the SV membrane (Pirazzini et al., 2014), where translocation of the L chain is expected to occur. Several inhibitors of the TrxR-Trx redox couple prevent the occurrence of the SNARE specific metalloprotease activity of the L chain of all serotypes of BoNTs, from A to G in cultured neurons. More importantly, these inhibitors largely prevent the BoNT-induced paralysis in mice in vivo, regardless of the serotype involved (Zanetti et al., 2015). The reduction of the single interchain disulfide bond is therefore a general and fundamental step of the BoNT [and TeNT (Pirazzini et al., 2013a)] mechanism of nerve terminal intoxication. The Trx tertiary fold is similar to that of ancestral chaperonins, so it is also possible that Trx functions as a chaperonin for L-chain translocation (Berndt et al., 2008; Dekker et al., 2011).
1.2.5 SNARE protein cleavage
The L chains of all known BoNTs are metalloproteases that are specific for one of the SNARE proteins: VAMP (vesicle-associated membrane protein; also known as synaptobrevin), SNAP-25 (synaptosomal-associated protein of 25 kDa) or syntaxin. BoNT/C cleaves both SNAP25 and syntaxin, BoNT/B, BoNT/D, BoNT/F and BoNT/G only target VAMP and BoNT/A and BoNT/E cleave SNAP-25. The fact that the inactivation of
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any one of these three proteins inhibits neurotransmitter release, is the strongest evidence that these three proteins form the core of the neuroexocytosis nanomachine (Sudhof and Rizo, 2011; Pantano and Montecucco, 2014). The SNARE family of proteins includes many isoforms of VAMP, SNAP-25 and syntaxin, which are differentially expressed in many non-neuronal cells and tissues. Although several of these isoforms can be cleaved by BoNTs, these substrates are not accessible in vivo, as non-neuronal cells lack appropriate receptors for the toxin (Schiavo et al., 2000; Binz, 2013).
With the exception of BoNT/A and BoNT/C, all BoNTs cleave isolated SNARE proteins by removing large cytosolic segments, which prevents the formation of the SNARE complex. BoNT/A and BoNT/C remove only a few residues from the C-terminus of SNAP-25 and this truncated form of SNAP-25 can form a stable SNARE complex (Hayashi et al., 1994), which is however unable to drive exocytosis. This suggest that cleaved SNAP-25 acts as a dominant negative factor in the function of a multimeric radial super-SNARE complex because the C-terminal segment is necessary for protein-protein interactions underpinning the formation of a radial SNARE super complex (Montecucco et al., 2005; Pantano and Montecucco, 2014). At the present stage, it cannot be excluded that SNAP-25 exists in different pools within the nerve terminal and that a sub-pool consisting of 10–15% of total SNAP-25 is the one involved in neuroexocytosis. It is also possible that the C-terminal segment of SNAP-25 inserts in the lipid bilayer, plays an essential role in membrane fusion (Pirazzini et al., 2017). The selectivity of BoNTs is shown by two examples that are relevant to the evolutionary biology of animal botulism. The replacement of a Gln with a Val at the P19 position of the VAMP cleavage site in TeNT, makes rats and chicken resistant to tetanus (Patarnello et al., 1993). BoNT/D is the most potent toxin on mice (lethal dose, 0.1ng/kg) but it is poorly toxic for humans (Eleopra et al., 2013) and rats, because one of their VAMP1 exosites is not conserved (Peng et al., 2014).
1.3 NEUROEXOCYTOSIS AND SNARE PROTEINS
Synapses can be of two forms: electric synapses and chemical synapses. A prototypical example of chemical synapse is represented by the neuromuscular junction (NMJ) that allows the transmission of a muscular nerve impulse, by the release of neurotransmitter in the synaptic space and inducing muscular contraction. In general,
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neurotransmitters are synthesized in the neuronal cytosol and are stored in the presynaptic nerve terminal inside small synaptic vesicles (SSV) (Jahn and Fasshauer, 2012). The accumulation of neurotransmitters in the lumen of synaptic vesicles is driven by the electrochemical proton gradient that is generated by the vesicular ATPase proton pump. This pump is located in the synaptic vesicle membrane and pumps protons into the synaptic vesicle using the energy that is released by ATP hydrolysis. The synaptic vesicles either form a reserve pool of neurotransmitters within the nerve terminal, because they are bound to the actin cytoskeleton via interactions regulated by the phosphorylation of proteins, such as the synapsins. Otherwise, they bind to specialized sites of the presynaptic membrane that are known as “active” zones (Harlow et al.,2013; Zhai and Bellen 2004), in a process known as Docking. This step leads to stabilization of the binding by additional protein-protein interaction involving a set of SNARE proteins (Sudhof and Rizo 2012; Kasai, Takahashi and Tokumaru 2012). In the subsequent step, which is known as priming, a series of proteins plays an important role, these are:
I. Two synaptic vesicle integral membrane proteins, VAMP (also known as synaptobrevin) and synaptotagmin (Syt), called V-SNARE;
II. Two proteins in the presynaptic membrane, SNAP25 and syntaxin, called T-SNARE;
III. Cytosolic protein including complexin and Munc-18.
The interaction between the V-SNARE and T-SNARE, forms the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex and enables the synaptic vesicle to fuse rapidly with the presynaptic membrane in response to Ca2+
influx (Fig 4). In particular, Syt interacts with presynaptic membrane inositol phospholipids, whereas VAMP forms a coiled-coil complex with SNAP-25 (25-kDa SNAP) and syntaxin, in a process that is regulated by Munc18 and other proteins. After docking, fusion is prevented by complexin, which functions as a brake, and together with Munc-18, also promotes the assembly of several SNARE complexes to form a radial super-SNARE complex (Pantano and Montecucco, 2014; Megighian et al., 2013). This is the “core” of the whole process that mediates neurotransmitter release. The carboxy terminus of SNAP-25 has an essential role in protein-protein interactions between the SNARE complexes within the super-SNARE complex. It is likely that the synaptic vesicle and the presynaptic membrane are hemifused in the primed state
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(Chernomordik and Kozlov, 2008), which would account for the ultrafast (milliseconds or less) release of neurotransmitter at the NMJ. The active zone is rich of Ca2+ channels
and the depolarization of the nerve terminal results in the opening of the Ca2+
channels and the influx of Ca2+ ions that induces the release of the primed synaptic
vesicle following binding to Syt. Such binding triggers a rapid conformational change that leads to a complete synaptic vesicle-presynaptic membrane fusion and the formation of a pore through which neurotransmitter is released into the synaptic cleft. Neurotransmitter diffuses out of the nerve terminal and binds to a postsynaptic receptor, which triggers signalling in the postsynaptic cell. In the case of the NMJ, acetylcholine (ACh) is released and binds to the acetylcholine receptor (AChR), which results in depolarization of the muscle plasma membrane, leading to Ca2+ entry and
muscle contraction. During neurotransmitter release, the lumen of the synaptic vesicle is transiently opened to the outside, but it is later internalized into the nerve terminal by endocytosis (Saheki and De Camilli, 2012). The exocytosis is rapidly (<1s) followed by endocytosis, and the two processes are strictly coupled: inhibition of one process leads to inhibition of the other. Most endocytosis of synaptic vesicles at the NMJ is mediated by a clathrin coat and of a GTP-dependent action of dynamin. After internalization and uncoating, the synaptic vesicle is refilled with neurotransmitter and the next cycle of neurotransmission begins.
Figure 4 Neurotransmission at synapses. Major phases and proteins implicated in exocytose and neurotransmitter release coupling with endocytosis. (Rossetto et al. 2014)
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1.4 MOLECOLAR TARGETS
The nine CNTs are remarkably specific proteases. Among the many proteins and synthetic substrates assayed so far, only three of them, the so-called SNARE proteins, have been identified (Table 1- Schiavo et al., 2000). TeNT and BoNT/B, /D, /F and /G cleave vesicle-associated membrane protein (VAMP)/synaptobrevin, but each at different sites. BoNT/A and /E cleave 25-kDa synaptosomal-associated protein (SNAP-25) at two different sites and BoNT/C cleaves both syntaxin and SNAP-25. Strikingly, TeNT and BoNT/B cleave VAMP at the same peptide bond (Gln-76-Phe-77), yet when injected in the animal, they cause the opposite symptoms of tetanus and botulism, respectively (Schiavo et al., 1992). This observation clearly demonstrated that the different symptoms derive from different sites of intoxication rather than from a different molecular mechanism of action of the two neurotoxins.
Table 1 botulism neurotoxins: target and peptide bond specificities
25 kDa Synaptosomal-associated-protein (SNAP-25)
SNAP-25 is a major palmitoylated protein in the CNS (Hess et al., 1992; Oyler et al., 1989; Scheer, 1989). Because of the absence of a canonical transmembrane segment (Fig. 5), SNAP-25 membrane localization is thought to be mediated by the palmitoylation of cysteine residues located in the middle of the polypeptide chain (Hess et al., 1992; Lanc and Lin, 1997). SNAP-25 is conserved from yeast to humans (Scheer et al., 1986), with little variation in length and size, it self-associates to form a disulfide-linked dimer, both in vitro and in vivo (Sadaul et al., 1997). The SNAP-25 forms a stoichiometric complex with the putative Ca2+ sensor synaptotagmin (Syt), and
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this interaction is believed to be important for the Ca2+-dependent phase of
neurotransmitter release (Banerjee et al., 1996). Furthermore, SNAP-25 was demonstrated to interact in a Ca2+-dependent manner with Hrs-2, an ATPase having a
negative regulatory effect on neuroexocytosis. SNAP-25 is required for axonal growth during neuronal development and in nerve terminal plasticity in the mature nervous system (Geddes et al., 1999). SNAP-25 is developmentally regulated, with the two isoforms A and B switching their expression in the nervous system and neuroendocrine cells at birth (Bark and Wilson, 1994). The synthesis of both isoforms is upregulated in hippocampal neurons during long-term potentiation, thus suggesting their involvement in synaptic plasticity (Roberts, 1998).
Figure 5 Schematic structure of SNAP-25. Cleavage sites for BoNT/A, /C and /E (arrows) and the two segments essential for the interaction whit other SNARE (grey boxes) are indicated. (Schiavo et al., 2000)
VAMP/Synaptobrevin
VAMP (also referred to as synaptobrevin) is a protein of 13 kDa localized to synaptic vesicle, dense core granules, and synaptic–microvesicles and is the prototype of the vesicular SNARE (V-SNARE) (Bennett and Scheller, 1994). Four functional domains can be distinguished in VAMP molecule (Fig. 6) (Trimble, Cowan and Scheller, 1988). The NH2-terminal 33-residues-long part is proline rich and isoform specific, and it is
involved in protein-protein interaction with others components of the neuroexocytosis apparatus and transport proteins. Instead, the following region (residues 33-96) is very well conserved through evolution and contains coiled-coil regions and sites of
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phosphorylation for the Ca2+/calmodulin- dependent protein kinase type II and the
casein kinase type II (Nielander et al., 1995). Moreover, this central portion of VAMP coils around complementary region of SNAP-25 and syntaxin in the SNARE complex and contains the site of interaction and cleavage by the CNT. The protein is anchored to the synaptic vesicle membrane via a single transmembrane domain, which is followed by a poorly conserved tail, protruding in the vesicle lumen, of variable length in different species. Splicing variants of VAMP-1 with modified COOH-terminal sequences have been described. In one case (VAMP-1B), the splicing process shortens the predicted transmembrane region by four residues and appends a functional mitochondrial localization signal to the COOH terminus of VAMP (Isenmann et al., 1998).
Figure 6 Schematic structure of VAMP. The cleavage sites for CNT are indicated by arrow. (Schiavo et al., 2000)
Ten different isoforms have been identified based on structural sequence similarity, but only three isoforms of VAMP have been extensively characterized: 1, VAMP-2, and cellubrevin (ubiquitous) (Baumert et al., 1989; McMahon et al., 1993; Trimble et al., 1989). The VAMP isoforms are present in all vertebrate tissues, but their relative amount and distribution differs (Majo et al., 1998). On the synaptic vesicle membrane, VAMP is associated with synaptophysin, a major component of SSV membrane and with subunits of the V-ATPase (Calakos and Scheller, 1994; Edelmann et al., 1995).
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VAMP-2, but not VAMP-1 or cellubrevin, interacts with a prenylated Rab acceptor via its proline-rich and its transmembrane segment as assessed by yeast two-hybrid screening and direct binding (Mandic et al., 1997).
Syntaxin
Syntaxin is a type II membrane protein of 35 kDa located mainly on the neuronal plasmalemma (Fig. 7). The NH2-terminal portion is exposed to the cytosol and it is
followed by a transmembrane domain and few extracellular residues (Bennett et al., 1992; Bennett and Scheller, 1994). The cytosolic region is composed by two domains characterized by distinct structural features. The NH2-terminal domain of monomeric
syntaxin (residues 1–120) consists of three long a-helices, which are likely to be involved in protein-protein interactions (Fernandez et al., 1998). The central portion (residues 180–262) enters in a four helix bundle structure upon interaction with the other members of the SNARE protein complex: coiling around complementary regions of VAMP and SNAP-25 (Poirier et al., 1998; Sutton et al., 1998). Botulinum neurotoxin type C (BoNT/C) cleaves within this part of syntaxin and compromise the functional pairing of the vesicle with the presynaptic membrane. The last one part is the transmembrane segment (TM), followed by a short extracellular COOH-terminal segment.
Figure 7 Schematic structure of syntaxin I. Cleavage site for BoNT/C (arrow) and the segment for the interaction with other SNARE (grey boxes) are indicated. (Schiavo et al., 2000).
Various syntaxin polymorphisms exist within the nervous tissue, and syntaxins constitute a protein family with more than 20 isoforms in mammals and with homologs in yeast and plants. In the active zones, syntaxin is associated with several types of Ca2+ channels (Bezprozvanny et al., 1995). Syntaxin is also present on
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chromaffin granules (Tagaya et al., 1995) and it undergoes, together with SNAP-25, a recycling process in organelles indistinguishable from synaptic vesicles (Walch-Solimena et al., 1995). Syntaxin interacts with the other T-SNARE SNAP-25 and the V-SNARE VAMP/synaptobrevin to form the synaptic V-SNARE complex, which represent the core of the neuroexocytosis apparatus. The SNARE complex formation is controlled by the interaction of syntaxin with Munc-18, and it is regulated by protein kinase C (Fujita-Yoshigaki et al., 1996). Syntaxin could have a role in synaptic plasticity because several isoforms, including syntaxins 1 and 3, undergo a complex pattern of alternative splicing and expression during long-term potentiation (Hicks et al., 1997). This polymorphism could be important for a selective interaction with specific Ca2+
channels and thus a direct modulation of Ca2+ entry and a distinct SNARE complex
formation with different SNAP-25 and VAMP isoforms.
1.5 DURATION OF ACTION
One remarkable aspect of the peripheral neuroparalysis induced by BoNTs, with respect to others toxins which kill cells, is its complete reversibility. Patients with botulism fully recover if death by respiratory paralysis is prevented by mechanical ventilation and supportive care is provided (Johnson and Montecucco, 2008). This is due to the fact that BoNTs are neither cytotoxic nor they cause any axonal degeneration, probably because the cell body is frequently located at distance away from the intoxicated terminal. The very extensive and long-term experience with BoNT/A1 and BoNT/B1 as therapeutics has provided no indications of neuronal damage after repeated treatments extended over many years (Naumann and Jankovic, 2004; Naumann et al., 2006). The BoNT duration of action is of paramount importance because it determines the duration of hospitalization of botulism patients and the duration of the therapeutic effects. These aspects of BoNTs biology and pharmacology are very relevant in the clinic and additional applications.
The duration of the BoNTs-induced neuroparalysis varies with: BoNT serotype, dose, animal species, mode of administration and type of nerve terminal. The order of duration of action in mice and humans is: BoNT/A1 ̴ BoNT/A2 ̴ BoNT/A5 ̴ BoNT/C1 > BoNT/A4 > BoNT/A4 > BoNT/B1 ̴ BoNT/D > BoNT/F1 ̴ BoNT/G >> BoNT/E1 (Foran et al., 2003; Eleopra et al., 2004; Keller, 2006). BoNT/D is poorly active in humans but very potent in mice (Eleopra et al., 2013). The duration of action of BoNTs is about
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three times longer in humans than in mice (i.e., BoNT/A 3–4 months versus 1 month, respectively), and skeletal muscles recover about three times faster than autonomic cholinergic nerve terminals (in humans 3–4 months versus 12-15 months for BoNT/A1) (Pirazzini et al., 2017). The major difference is that the BoNT/A1 paralyzed skeletal terminal undergoes a process of sprouting guided by the perisynaptic Schwann cells (Son and Thompson, 1995; Meunier et al., 2002). Sprouts originate from the unmyelinated nerve terminal and reach other muscle fibres as well as the same fibre in another position. Eventually, sprouts degenerate and the function of the original endplate is re-established. From these observations, it appears that the sprouting process influences the lifetime of the BoNT L domain, explaining why duration is long where there is no sprouting (e.g. neurons in culture, autonomic terminals) and about four times shorter where sprouting takes place (i.e. at the NMJ).
The main factor that contributes to the biological action of BoNTs is the L chain half-life, although it is very difficult to estimate in vivo. In fact, the main studies were performed in vitro by transfection techniques (Fernandez-salas et al., 2004a) for BoNT/A1 and BoNT/E1 L chain, the longest and the shortest respectively, in vivo. It appears that BoNT/A1 L chain has a longer lifetime than that BoNT/E1 because it escapes form the action of the cell degradation system (Tsai et al., 2010). In fact, BoNT/E L chain is ubiquitinated and targeted to the ubiquitin-proteasome system, whereas BoNT/A1 L chain appeared refractory to this degradation pathway. This resistance may come from the ability of the L chain of BoNT/A1 to recruit deubiquitinases, specialized enzymes that remove polyubiquitin chains.
It is relevant that the cleavage of 10–15% of SNAP-25 was found to be sufficient to cause a complete blockage of neurotransmitter release (Keller et al., 2004) and that BoNT/E, removes a large part of the C-terminal half of SNAP-25, whereas BoNT/A and BoNT/C removes only nine and eight residues from the C terminus, respectively. The explanation of these results is that there are different pools of SNAP-25 within nerve terminals and that only a 10–15% of total SNAP-25 is actively involved in neurotransmitter release and accessible to the proteolytic action. Furthermore, cleaved SNAP-25 may contribute significantly to the long duration of action of BoNT/A by acting as a dominant negative component of a neuroexocytosis nanomachine, that
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is critically dependent on an intact SNAP-25 C-terminus (Megighian et al., 2013; Pantano and Montecucco, 2014).
1.6 CLINICAL USES OF BoNTs
The demonstration that the inhibition of the nerve-muscle impulse is followed by a functional recovery of the NMJ provides the scientific basis of the rapidly growing use of BoNT in the therapy of a variety of human diseases caused by hyperfunction of cholinergic terminals (Jankovic and Hallett, 1994; Scott, 1989). Injection of minute amounts of BoNT/A into the diseased targets robustly reduces nerve terminal activity, whit long-lasting symptom relief (Davletov et al., 2005; Johnson 1999; Montecucco et al., 2005; Hallett 2013). BoNT/A is generally used, but other BoNT types are currently under clinical trial. Injection of BoNT is currently recognized as the best available treatment for dystonias and for certain types of strabismus (one of the first treatment carried out Alan Scott and Edward Schantz), and it is now being extended to several other human pathologies (Jankovic and Hallett, 1994). In 1989, the Food and Drugs Administration (FDA) approved BoNT/A for the treatment of strabismus, blepharospasm and hemicefalic spasm. Since then, the number of indication being treated has increased quickly and now include numerous many other disorders (table
2), which were previously refractory to conventional treatments as the treatment of
the pain and depression. This treatment can be repeated several times, without major side effects such as the development of an immune response. If anti-neurotoxin antibodies are produced, treatment can be continued with another BoNT serotype.
1.6.1 Dystonias
Dystonias are a heterogeneous group of disorders characterized by sustained involuntary muscle contractions, frequently causing repetitive twisting movements, abnormal postures, and pain (Albanese and Lalli, 2009, 2012). Dystonias are classified by clinical characteristics such as body distribution, nature of the symptoms and associated features or cause. In the first case, we distinguish:
1) Generalized, when interests is the majority of the body or all the body. For example dystonia musculorum deformans (Oppenhiem, Flatau-Sterling syndrome)
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2) Focal, when is affects a muscle or a group of muscles in specific parts of the body. For example the blepharospasm.
3) Multifocal, when are affected more than one parts of the body
4) Segmental, affect two adjoining parts of the body, frequently involving the legs and back.
TABLE 2 Therapeutic uses for botulinum neurotoxin
Ophtalmology Strabismus (a,b,c) Nistagmus Neurology Focal Dystonias Blepharospasm (a,b,c)
Cervical dystonia (a,b,c) (Torticollis, anterocollis, laterocollis) Occupational dystonias (writer’s cramp (b), musician’s cramps) Laryngeal Dysphonia (c)
Oromandibular dystonia Lingual dystonia Nondystonic disorders Hemifacial spasm (a,b,c)
Tremor (essential, parkinsonism) Tics
Bruxism
Spasticity (poststroke, multiple sclerosis, brain or spinal cord injury) Focal spasticity (a,b,c): Upper and lower limb spasticity Nonfocal: hemispasticity, paraspasticity, tetraspasticity Cerebral palsy (a,b)
Hyperhidrosis (a,b,c)
Focal: axillary, palmar, plantar Diffuse
Hypersalivation
Sialorrhea (b) (motoneuron diseases/amyotrophic lateral sclerosis) Drooling (b) (Parkinsonian syndromes)
Frey’s syndrome/gustatory sweating Aesthetic (muscle)
Glabellar rythides (a,b,c)
Pain
Muscular Dystonia Spasticity
Chronic myofascial pain Temporomandibular disorders Low back pain
Nonmuscular
Migraine (chronic (a) and tension type migraine) Neuropathic pain
Trigeminal pain Pelvic pain
Urology
Detrusor sphincter dyssynergia
Overactive bladder (a,b,c) (Idiopathic or neurogenic detrusor overactivity) Urinary retention
Bladder pain syndrome Pelvic floor spasms
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Gastroenterology
Achalasia
Chronic anal fissures
Psychiatry
Depression (d)
(a) USA approved indication; (b) EU approved indication; (c) Evidence-based therapeutic indication; (d) To be evaluated. (Pirazzini et al., 2017)
Localized injections of BoNT provide a transient symptomatic relief in focal dystonia syndromes, as demonstrated by several randomized controlled studies and by a large number of uncontrolled studies (Hallett et al., 2013; Guidubaldi et al., 2014). In general, the therapeutic benefits of BoNT in dystonias derive from a decreased acetylcholine release from alpha motoneurons at the NMJ and the ensuing relaxation of the affected skeletal muscle. However, substantial evidence suggests that BoNT injected peripherally may also influence indirectly CNS function by the inhibition of intrafusal muscle fibers due to the blocking of the gamma motoneurons (Giladi, 1997; Hallett, 2000; Rosales and Dressler, 2010). A form of focal dystonia is blepharospasm which involves the periocular muscles: their contraction produces forced eyelid closure, sometimes leading to functional blindness. FDA approved BoNT/A1 as treatment for blepharospasm in 1989. Response rates of around 90% make blepharospasm one of BoNT’s most successful indications. Adverse effects are usually mild and always transient and include local hematoma, ptosis, and diplopia (Dressler, 2012).
1.6.2 Spasticity
Spasticity describes the combination of a central paresis together with various forms of muscle hyperactivity, including dystonia, rigidity, and spasms often associated with pain. Most frequent etiologies include cerebral stroke, multiple sclerosis, traumatic brain injury, spinal cord injury and infantile cerebral palsy. The goal of spasticity treatment is to reduce motor overactivity to improve movement without worsening weakness. BoNT treatment of spasticity can reduce muscle tone, improve function, facilitate nursing and prevent contractures and decubitus. BoNT therapeutic effects are better established for spasticity in the upper rather than lower limb (Esquenazi et al., 2013). It usually involves large BoNT doses (typically 300–500 U Botox or Xeomin or 600– 1000 U Dysport for arm or leg spasticity) without signs of systemic toxicity (Dressler et al., 2015). A higher dilution with larger injection volumes show higher
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degree of paralysis, probably because of larger spreading to neuromuscular junctions remote from injection site (Gracies et al., 2009).
1.6.3 Pain
Studies conducted on urologic disorder, about the pathophysiology of the overactive bladder, sees BoNT acting primarily by inhibiting acetylcholine release from parasympathetic nerve endings inducing detrusor muscle relaxation. In addition BoNT/A1 reduces the release of glutamate, of substance P and of calcitonin gene-related peptide from the peripheral terminals of afferent bladder neurons (Duggan et al., 2002; Rapp et al., 2006). The BoNT inhibition of secretion of these neuropeptides and ATP as well as cyclooxygenase-2 products, which are inflammatory response mediators released from nociceptive sensory endings in response to noxious stimuli (Khera et al., 2004; Chuang et al., 2008), explain the therapeutic benefit of BoNT injection in painful bladder conditions (Smith et al., 2004; Wang et al., 2016). It is particularly interesting that the analgesia provided by BoNT injection occurs before muscle paralysis and outlasts any muscle weakness. It is now well documented that the analgesic effects of BoNT/A1 are related not only to its paralytic effect, but also to an effect on the nociceptor system (Wheeler and Smith, 2013). The antinociceptive effect of BoNT/A1 would be primarily mediated by the blockade of neuropeptides and inflammatory mediators release, and by the inhibition of plasma membrane exposure of pain sensors at peripheral level. Indeed, in cultured sensory neurons, the TNF-alpha induced surface trafficking of TRPV1 and TRPA1 channels is mediated by SNAP-25, VAMP-1 and syntaxin-1 and it is inhibited by the serotypes of BoNTs that selectively cleave their respective SNAREs (Meng et al., 2014, 2016). Within the pain field, only chronic migraine is an approved FDA indication for BoNT/A1. All other areas are currently considered off label, probably for the lack of standardized guidelines for BoNT application and dosage for pain management, and the lack of appropriate definition of study primary outcomes.
1.7 MEASURING BoNT/A CENTRAL EFFECT IN HUMANS
The cleavage of SNAP-25 by the BoNT activity leads to the chemical denervation result in weakness of the striated muscles, thus reducing the pathological, involuntary
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muscle contractions. Since the introduction of BoNT/A to treat various disordered associated with increased muscle tone, it has become clear that clinical effects cannot be exclusively explained by peripheral neuroparalysis. In literature there are several reports of: (i) clinical benefit exceeding the duration of peripheral chemo-denervation, (ii) reduction in the severity of spasticity in spite of little neuromuscular blockade and (iii) improvement of function in muscles acting as antagonists to the injected one. All these data are indeed consistent with a central action of the neurotoxin.
At least three mechanism have been postulated by which peripherally injected BoNT/A, may influence activity in central circuits. The first one is BoNT/A- mediated blockade of gamma motor endings in the muscle, whit the consequent reduction of spindle afferent (Ia fibers) input from the injected muscle (Filippi et al., 1993). This may indirectly change the excitability of spinal pathways as well as cause transitory alterations of motor maps at cortical level. A second possibility is the blockade of the neuromuscular connection between alpha-motoneuronal endings and extrafusal muscle fibers, which may lead to adaptive reorganization in the motoneuron (i.e., plasticity induced by peripheral denervation). The last one, retrograde axonal transport to spinal cord sensory/motor neurons and transcytosis to afferent synapses with consequent central chemo-denervation. This allows for direct central effects of BoNT/A (Mazzocchio and Caleo, 2014). In the case of migration along motor axons, BoNT/A action could occur at the cholinergic synapse between motoneurons and Renshaw cell through anterograde transport or the inhibitory Renshaw cell synapse on motoneuron dendrites through transcytosis. Whichever mechanism should lead to a reduction in the recurrent inhibition of spinal motoneurons. The effect of BoNT/A, at doses routinely used for treating leg spasticity, was tested from one injected muscle (soleus) to motoneurons supplying an untreated muscle, distant from the injection site (quadriceps). Stimulation parameters (producing recurrent inhibition, mediated by Renshaw cells) were monitored on a third non-injected muscle (flexor digitorum brevis) but innervated by the same nerve as the soleus (tibial nerve). One month after BoNT/A injection in the soleus muscle, the level of recurrent inhibition in quadriceps was depressed on average by about 46% of its level before BoNT/A injection in the soleus muscle (Mazzocchio and Caleo, 2014). All this is explained by a decrease global pool activity of Renshaw cells. Similarly, in animals, depressed recurrent inhibition
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after BoNT/A muscle injection was observed when the global activity of Renshaw cells was tested (Wiegand and Wellhoner, 1977). These results were explained by an action of BoNT/A on the inhibitory synapses between the Renshaw cells and the motoneuron. This mechanism can be only possible through retrograde axonal transport to the motoneuron cell body and trancytosis, like for tetanus neurotoxin. Evidence for reduced motor axon–Renshaw cell activity after peripheral BoNT/A injection comes from morphological and physiological studies in the rat (Clowry et al., 2006; Gonzalez-Forero et al., 2005; Sanna et al., 1993). Changes in motoneuron intrinsic properties, number of recurrent collaterals and strength of synaptic contact starting 1 to 3 weeks after BoNT/A intramuscular injection have also been observed in the animal and could cause a general reduction in the excitability level of motoneurons likewise affecting all their targets (Caleo et al., 2009).
1.8
BoNT/A TRANSPORT: EXSPERIMENT IN THE RAT VISUAL SYSTEM
Uptake of BoNT/A at the nerve terminal occurs via synaptic vesicle endocytosis, followed by translocation of the L chain into the neuronal cytosol and cleavage of the target substrates (Binz and Rummel, 2009). While most of BoNT/A remains at the injection site, there is experimental evidence in animal models that this toxin can undergo retrograde axonal transport and transcytosis, particularly when high doses are used (Habermann, 1974; Wiegand et al., 1976; Moreno-Lopez et al., 1997; Antonucci et al., 2008b). Transcytosis refers to the process by which a ligand penetrates the neuron at one side, followed by its movement and release at the opposite end, with possible uptake by second-order neurons (Restani et al., 2011). This cell-to-cell trafficking is important because it may allow the toxin to exert its actions at a distance from the injection site. At the Neuroscience Institute laboratory in Pisa, a series of experiments have been performed which have demonstrated both retrograde propagation and anterograde propagation of BoNT/A, from the injection site (Antonucci et al. 2008; Restani et al., 2011, 2012).
1.8.1 Anterograde propagation
To examine anterograde propagation of BoNT/A effects in the visual system, BoNT/A was injected into the vitreous humor of adult rats. Three days later, retinal and tectal
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sections were immunostained with an antibody specific for BoNT/A-truncated SNAP-25. In the injected retina, strong staining was found in the inner plexiform layer; cell bodies of bipolar cells in the inner nuclear layer were also stained. A fainter labeling appeared in cell bodies of RGCs (Retinal Ganglion Cell) and in the optic nerve fiber layer. To determine whether BoNT/A-truncated SNAP-25 is detectable in RGC axons, BoNT/A was injected intraocularly and the optic nerve was crushed to allow accumulation of the cleaved product. Labeled fibers were clearly evident on the side proximal to the crush 24 h after surgery, indicating presence of BoNT/A-truncated SNAP-25 within retinal axons. No specific staining was found in the contralateral, uninjected optic nerves and retinas. Examination of tectal sections revealed consistent labeling of the retinorecipient layers of the superior colliculus (SC) contralateral to the injected eye. Labeling was evident 3 days after injection and became particularly prominent at 15 days. Staining for cleaved SNAP-25 was typically stronger in the medial part of the SC, topographically corresponding to the injection site in the ventral retina. Labeling was virtually undetectable in the ipsilateral SC. Thus, injection of BoNT/A into the eye leads to the appearance of BoNT/A-truncated SNAP-25 in the contralateral tectum. This is consistent with the fact that in the rat, about 97% of the RGCs project contralaterally (Cowey and Perry, 1979). Labeling for BoNT/A-altered SNAP-25 also appeared in other retinorecipient nuclei such as the lateral geniculate nucleus and pretectal area 3–15 days after injection.
Anterograde propagation of BoNT/A effects requires axonal transport. To block microtubule-dependent transport, intraocular injections of the depolymerizing agent colchicine were used (Caleo et al., 2000, 2003; Antonucci et al., 2008). Injection of colchicine blocked the anterograde propagation of BoNT/A effects from the retina to the contralateral SC. The selective appearance of BoNT/A-truncated SNAP-25 in the SC contralateral to the injected retina, and the blockade of this anterograde transfer by colchicine strongly argue against a systemic spread of toxin effects (via blood or cerebrospinal fluid - CSF). To determine whether cleaved SNAP-25-positive profiles correspond to synaptic terminals, double-label immunofluorescence for BoNT/A-altered SNAP-25 and the synaptic vesicle marker synapsin was performed. The quantitative analysis demonstrated a very significant colocalization of cleaved SNAP-25 and synapsin immunoreactivity. To gain more insight into the type of nerve terminals
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containing truncated SNAP-25, double-label immunostaining for markers of excitatory (vGlut-1 and -2) and inhibitory synapses (vGAT) was performed. In the rat SC, vGlut-2 is specifically expressed by terminals of RGCs (Fujiyama et al., 2003; Caleo et al., 2009). Cleaved SNAP-25 displayed significant colocalization with vGlut-1 and vGAT while there was no colocalization with the retinal terminal marker vGlut-2. These immunostaining data suggest that cleaved SNAP-25 is not present within RGC terminals. To explore more directly whether cleaved SNAP-25 is present in RGC fibers, the authors took advantage of the fact that cleaved SNAP-25 associated with retinal terminals would be rapidly lost after section of the optic nerve, due to Wallerian degeneration of transected fibers. Therefore, BoNT/A was injected unilaterally into the vitreous in a group of rats, and allowed 3 days for anterograde transport to the SC. Then, the optic nerve corresponding to the injected eye was cut in a subset of the animals. The remaining animals were left unoperated. The authors allowed another 7 days for Wallerian degeneration of transected RGC fibers and then performed immunoblotting for cleaved SNAP-25 in tectal protein extracts (contralateral to the injected retina) in control and optic nerve sectioned rats. BoNT/A-truncated SNAP-25 was not decreased in the denervated tectum (control vs denervated).
In a second protocol, the authors used bilateral, intravitreal injections of BoNT/A, followed by section of the left optic nerve 3 d later and immunohistochemistry for cleaved SNAP-25 at 10 days. This protocol allows one to compare levels of BoNT/A truncated SNAP-25 in the denervated and control SC of the same animal. The authors found that staining for cleaved SNAP-25 was not abolished in the deafferented tectum. Quantitative analysis of the immunostaining revealed no differences between levels of BoNT/A-truncated SNAP-25 in the SC contralateral and ipsilateral to optic nerve section. Importantly, quantification of the retinal fiber marker vGlut-2 indicated a very robust decrease of the immunostaining demonstrating that optic nerve section effectively removes most of retinal terminals from the contralateral SC. The lack of presence of BoNT/A-truncated SNAP-25 in RGC fibers suggests a process of anterograde transport and transcytosis, by which BoNT/A (and/or its cleaved substrate) is released from RGC terminals and subsequently concentrates in other synaptic terminals in the tectum.
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1.8.2 Retrograde propagation
Application of BoNT/A into the rat optic tectum results in retrograde axonal transport with SNAP-25 cleavage in synaptic terminals of amacrine cholinergic neurons impinging on retinal ganglion cells. Moreover, it was examined whether BoNT/A can undergo additional cycles of retrograde transport and transcytosis that further expand the toxin spread away from the injection site. Between three to fifteen days after BoNT/A injection into the tectum, rod bipolar cells and phoreceptors, that are at least two synapses away from the injection site, were immunoreactive for BoNT/A cleaved SNAP-25. Stained photoreceptors were unambiguously identified by their location within the outer nuclear layer (ONL) of the retina. A subset of rod bipolar cells (identified by their specific marker protein kinase C) were also immunopositive for BoNT/A truncated SNAP-25. Muller glial cells were not labelled, ruling out aspecific, non-synaptic uptake of the toxin. No staining was found in the retina of untreated animals or animals injected with BoNT/A into the frontal cortex, indicating that retinal effects are not secondary to systemic spread of BoNT/A via the blood or cerebrospinal fluid. Thus, BoNT/A-induced SNAP-25 cleavage is mainly detected in amacrine cholinergic cells, but can also be found at least two synapses away from the injection site.
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2 AIM OF THE THESIS
The aim of this thesis is to investigate on transcytosis of botulinum neurotoxin type A (BoNT/A) and to clarify some aspects of its action on the central nervous system. After peripheral injection in the whisker pad of rats and mice, I examined its effects on the central areas of the facialis nucleus to which they project, in order to evaluate:
(i) retrograde transport and its kinetics in the catalytically active form (ii) the ability to undergo transcytosis and affect neurons of second order (iii) identify the specificity of transcytosis on cholinergic synapses.
