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Pirazzini, Marco (2013) The entry of tetanus and botulinum neurotoxins into neurons. [Tesi di dottorato]

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Abstract (inglese)

Tetanus and botulinum neurotoxins cause neuroparalysis by inhibiting neuroexocytosis. They are composed by two main chains: the 100 kDa heavy chain (H) mediates the neurospecific binding to target cells and chaperons the entry of the 50 kDa light chain (L). After binding on the plasma membrane, these neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, as shown here for the first time by immuno-electron microscopy. The lumenal acidic pH induces a structural change of the neurotoxin molecule that becomes capable of translocating its L chain into the cytosol, via a transmembrane protein-conducting channel made by the H chain. This is the least understood step of the intoxication process primarily because it takes place inside vesicles within the cytosol. In the present study, we describe how this passage can be made accessible to investigation by making it to occur at the plasma membrane of neurons. The neurotoxin, bound to the plasma membrane of cerebellar granular neurons in the cold, was exposed to a low pH extracellular medium and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. We found that the neurotoxin has to be bound to the membrane via at least two anchorage sites in order for a productive low-pH induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT) /B, /C and /D is similar and takes place in the same slightly acidic range, which comprises that present inside synaptic vesicles. Thanks to this reliable method we have also studied the temperature dependence and the time course of TeNT, BoNT/C and BoNT/D L chain entry across the plasma membrane. The time course of translocation of the L chain varies for the three neurotoxins, but remains in the range of minutes at 37 °C, whilst it takes much longer at °20 C. BoNT/C does not enter neurons at 20 °C. Translocation also depends on the dimension of the pH gradient. These data are discussed with respect to the contribution of the membrane translocation step to the total time to paralysis and to the low toxicity of these neurotoxins in cold-blood vertebrates.
Another fundamental event along CNTs neuron intoxication process is the reduction of the interchain disulphide bond. This is a conditio sine qua non to free the catalytic part of the molecule in the cytosol of neurons. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-Thioredoxin reductase-Thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins.
BoNT/A is the most frequent cause of human botulism and at the same time is largely used in human therapy. Some evidences indicate that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been formally proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscopy, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor SV2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle transmembrane pH gradient, we show that translocation of the neurotoxin into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by one or two neurotoxin molecules.
Another important aspect regarding CNTs research is their employment in human therapy. Accordingly, BoNT/A is almost invariably used in the treatment of many human diseases characterized by hyperactivity of peripheral cholinergic nerve terminals. Unfortunately, some patients are or become resistant to it. This drawback can be overcome by using other botulinum toxins, and pre-clinical studies have been performed with different toxin serotypes. Botulinum neurotoxin type D has never been tested in human muscles in vivo. Here we show that BoNT/D is very effective upon injection in mice, on the mouse hemidiaphragm preparation and on different rat primary neuronal cultures. From these experiments, doses to be injected in human volunteers were determined. The effect of the injection into the human Extensor Digitorum Brevis muscle was assayed by measuring the compound muscle action potential at different times after injection. Botulinum toxin type D was found to be very uneffective in inducing human skeletal muscle paralysis. These results are interpreted in terms of recent reports on neuronal surface receptors of this neurotoxin and of the established double receptor model of binding.

Abstract (italiano)

Le Neurotossine clostridiali (CNT), sono esotossine di origine batterica che causano le due note sindromi neuroparalitiche tetano e botulismo attraverso il blocco della neuroesocitosi. Sono composte da due catene principali legate covalentemente da un unico ponte disolfuro. La catena pesante di 100 kDa (H) fornisce il legame neurospecifico e media l'ingresso della catena leggera (L) di 50 kDa nei neuroni bersaglio. Dopo il legame sulla membrana plasmatica, queste neurotossine entrano nei terminali nervosi all'interno di vescicole sinaptiche tramite endocitosi. Qui il pH acido induce un cambiamento strutturale della molecola che diventa capace di traslocare la catena L nel citosol, grazie ad un canale predisposto dalla catena H. Questo è il passaggio meno conosciuto lungo tutto il processo perlopiù a causa della sede dove avviene, ovvero piccoli compartimenti endocitici scarsamente manipolabili dall’esterno. Nel presente studio si descrive come questo passaggio sia stato reso accessibile all'indagine facendolo verificare sulla superficie dei neuroni. La neurotossina, legata a freddo alla membrana plasmatica di neuroni primari di cervelletto, è stata esposta ad un mezzo tamponato a basso pH per simulare quanto avviene nelle vescicole sinaptiche. L'ingresso della catena L è stato monitorato misurando l'attività metalloproteasica specifica con un metodo raziometrico. Abbiamo trovato che la neurotossina deve essere necessariamente legata alla membrana con almeno due siti di ancoraggio al fine di andare incontro ad un cambiamento strutturale funzionale alla traslocazione. Inoltre, questo processo può avvenire solo se il disolfuro intercatena è intatto. La pH-dipendenza del riarrangiamento conformazionale della tossina tetanica (TeNT) e delle tossine botuliniche (BoNT) B, C e D è simile e avviene nello stesso intervallo, in una condizione di media acidità, tuttosommato simile a quella che si pensa esistere all'interno vescicole sinaptiche.
Grazie a questo affidabile metodo di indagine, abbiamo proceduto studiando la dipendenza dalla temperatura e la cinetica della traslocazione di TeNT, BoNT/C e BoNT/D. A 37 °C, la traslocazione delle tre diverse tossine varia nel tempo, ma rimane sostanzialmente nell'intervallo di minuti minuti, mentre ne richiede molto più a 20 °C. BoNT/C non trasloca a 20 °C. La traslocazione, come precedentemente visto, dipende anche dalla dimensione del gradiente di pH. Questi dati vengono discussi considerando l’intero arco di tempo necessario alla tossine per intossicare i neuroni, così come la scarsa tossicità delle tossine nei vertebrati a sangue freddo.
Un altro evento fondamentale lungo il processo di intossicazione è la riduzione del legame disolfuro intercatena. Questa è una conditio sine qua non per liberare la parte catalitica della molecola nel citosol dei neuroni. Utilizzando inibitori specifici dei diversi sistemi ossidoreduttivi citoplasmatici, si è dimostrato che il sistema NADPH-tioredossina reduttasi-tioredossina, è il principale responsabile di questo evento. Inoltre, auranofin viene indicato come possibile molecola lead per la progettazione di nuovi inibitori di queste neurotossine.
BoNT/A è responsabile della maggior parte dei casi di botulismo nell’uomo e allo stesso tempo è indicata, quasi senza alternative, come agente terapeutico per il trattamento di numerose condizione patologiche. Alcune prove indicano che essa penetra all’interno dei terminali nervosi via endocitosi di vescicole sinaptiche, ma questo non è mai stato formalmente provato. La subunità catalitica accede quindi al citosol grazie ad un cambiamento conformazionale guidato da un gradient di pH. Tuttavia, quale sia il compartimento acido sfruttato dalla tossina per innescare il cambiamento conformazionale non è stato ancora determinato. Attraverso esperimenti di immuno detezione e miscroscopia elettronica, abbiamo dimostrato che BoNT/A sfrutta il riciclo di vescicole sinaptiche per essere internalizzata e che il numero massimo di tossine per vescicola è dettato dal numero di recettori proteici, nella fattispecie SV2, presenti all’interno della stessa, piuttosto che dal recettore glicolipidico presente sulla membrane esterna. A suffragio di ciò, la rapida inibizione dell’acidificazione dei compartimenti acidi attraverso specifici inibitori, mostra una cinetica di traslocazione di BoNT/A talmente rapida da poter escludere con certezza che tale evento possa avvenire a livello di organelli acidificabili diversi dalle vescicole sinaptiche, quali possono essere ad esempio gli endosomi. Presi nel loro insieme, questi risultati propendono per un funzionamento di BoNT/A come una nanomacchina capace di traslocare la subunità catalitica attraverso l’impiego di una o tuttalpiù due molecole.
Un altro aspetto per cui la ricerca sulle CNT è importante è sicuramente il loro impiego in terapia umana. BoNT/A è usata nel trattamento di molte malattie umane caratterizzate da iperattività dei terminali nervosi periferici colinergici. Purtroppo, alcuni pazienti sono o diventano resistenti alla terapia. Questo inconveniente può essere superato utilizzando altre tossine botuliniche ed infatti studi pre-clinici sono stati condotti con differenti sierotipi di tossina. La neurotossina botulinica di tipo D non è mai stato testata in vivo su muscoli umani. Qui viene mostrato che BoNT/D rappresenta la tossina più efficace in preparazioni in vivo ed ex vivo nei topi. Da questi esperimenti preliminari, sono state determinate le dosi da testare in volontari umani. L'effetto della iniezione nel muscolo delle dita umano Extensor Digitorum Brevis è stata saggiata misurando la risposta del potenziale d’azione evocato nei muscoli a diversi tempi dopo l'iniezione. BoNT/D è risultata essere scarsamente efficace nella neuroparalisi del muscolo scheletrico umano ed è per cui poco appetibile per l’uso umano. Questi risultati sono stati interpretati considerando i recenti risultati in merito ai recettori sfruttati da tale sierotipo e il modo con cui la stessa si lega ad essi.

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Tipo di EPrint:Tesi di dottorato
Relatore:Montecucco, Cesare
Dottorato (corsi e scuole):Ciclo 25 > Scuole 25 > BIOSCIENZE E BIOTECNOLOGIE > BIOLOGIA CELLULARE
Data di deposito della tesi:29 Gennaio 2013
Anno di Pubblicazione:29 Gennaio 2013
Parole chiave (italiano / inglese):Botulinum neurotoxins, Tetanus neurotoxin, translocation domain, interchain disulphide, neuromuscular junction
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/04 Patologia generale
Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Biomediche
Codice ID:5673
Depositato il:22 Ott 2013 12:11
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