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Carli, Luca (2009) Studio degli effetti indotti dalle tossine botuliniche a livello di muscolo scheletrico. [Tesi di dottorato]

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Abstract (inglese)

The first project of this thesis is focalized on the study of muscle effects induced by Botulinum neurotoxins.
On the basis of the specific myosin heavy chain isoform that they express, mammalian skeletal muscle fibers are classified as Slow (type I) or Fast (type 2) fibers. Slow fibers show low velocity of shortening and high fatigue resistance, whereas fast fibers show high velocity but lesser resistance. Muscle fibers composition is strongly influenced by nerve activity in adult skeletal muscle; in fact, a reduced neuromuscular activity (for example, spinal cord injury) promotes a slow-to-fast fiber transformation in muscle, whereas an increase of muscle activity cause a shift in the opposite direction (fast-to-slow).
We observed that a blockade of nerve activity, induced by Botulinum Neurotoxin type A (BoNT/A), promotes the expression of the slow isoform of myosin heavy chain (MyHC) in contrast with other neuromuscular inactivity models. In order to correlate the MyHC isoform switch with the muscle fiber denervation by BoNT injection we analysed the expression of the marker N-CAM (neural cell adhesion molecule). N-CAM is an integral membrane glycoprotein which accumulates on muscle fiber membrane after denervation and it is not expressed in muscle under physiological condition. The results obtained show that BoNT induced-slow fibers never express N-CAM, while nearby fibers of the same muscle are positive for N-CAM. This observation would suggest that these fibers are still active and that they could undergone to a compensatory overload that lead them to a slow phenotype shift. Current experiments are performed to better understand this phenomenon and to investigate if botulinum neurotoxins block preferentially some motor units rather than others.
The second project is based on the study of diffusion of different Botulinum neurotoxin type A formulations injected in the mouse leg.
Botulinum neurotoxin type-A is very effective in the therapy of a wide range of human syndromes characterized by hyperactivity of peripheral cholinergic nerve terminals. Little diffusion of this toxin from the site of injection is commonly observed, but even minor changes in this property would greatly affect the validity of the treatment. Different pharmacological formulations of botulinum toxin type A (BoNT/A) are available and they may have different diffusion characteristics due to protein complex size, product format and pharmacological properties.
Here, we have assessed the extent of diffusion of three commercial preparations of botulinum neurotoxin type A: Botox? (Allergan, Inc.), Dysport? (Ipsen, Ltd.) and Xeomin? (Merz Pharmaceuticals), using a novel and highly sensitive test based on Neural Cell Adhesion Molecule (N-CAM) expression in muscle. N-CAM is a membrane glycoprotein which accumulates on muscle fibres after denervation and is not expressed in the untreated adult muscle. This allows a fine monitoring of the functional diffusion of this toxin and the sensitivity of the present assay is emphasized by the use of the mouse model because of the small muscle dimensions. The results presented here indicate that there is no significant difference between Botox?, Dysport? and Xeomin? with respect to diffusion into adjacent muscles in the mouse leg.

Abstract (italiano)

La prima parte di questo lavoro riguarda lo studio degli effetti indotti dalle tossine botuliniche nel muscolo scheletrico.
In base a diverse caratteristiche, le fibre costituenti il muscolo scheletrico vengono distinte in fibre lente e fibre veloci. Le fibre muscolari lente (tipo I) presentano bassa velocità di contrazione ed elevata resistenza all’affaticamento. Le fibre veloci (tipo II) presentano elevata velocità di contrazione ma bassa resistenza all’affaticamento. E’ stato dimostrato da diversi studi che stati di inattività muscolare portano a una maggiore espressione di fibre veloci. Al contrario, l’aumento dell’attività muscolare induce un incremento in fibre lente. Le nostre osservazioni riportano, contrariamente a quanto atteso, che in seguito alla paralisi indotta da tossina botulinica una parte delle fibre del muscolo tibiale anteriore (che fisiologicamente presenta fibre veloci) cambiano il loro fenotipo diventando lente. Per correlare il cambiamento fenotipico delle fibre con la paralisi indotta dalle BoNTs è stata analizzata l’espressione di N-CAM (neural cell adhesion molecule). N-CAM è una glicoproteina di adesione che viene espressa sulle fibre muscolari in seguito a denervazione o paralisi mentre non è espressa in condizioni fisiologiche. I risultati ottenuti dimostrano che le fibre diventate lente non esprimono mai N-CAM mentre le altre fibre dello stesso muscolo sono positive per tale marcatura. Queste osservazioni suggeriscono che tali fibre siano andate incontro ad un sovraccarico compensatorio che le ha portate al cambiamento da fibre veloci a fibre lente. Gli esperimenti attualmente in corso sono rivolti a comprendere tale fenomeno e capire se le tossine botuliniche possano essere selettive per certe unità motorie rispetto ad altre.
La seconda parte del progetto sfrutta la proprietà di N-CAM di essere espressa solo nelle fibre muscolari paralizzate di topo adulto per lo studio comparativo della diffusione di diverse preparazioni commerciali di tossina botulinica di sierotipo A.
Molti disturbi dovuti ad iperattività colinergica del sistema nervoso periferico vengono comunemente trattati nella pratica clinica mediante l’uso di preparazioni farmaceutiche di tossina botulinica. In questo progetto, tre preparazioni commercialmente disponibili di tossina botulinica di sierotipo A (Botox?-Allergan, Inc., Dysport?-Ipsen, Ltd. e Xeomin?-Merz Pharmaceuticals), sono state studiate e comparate in termini di diffusione dal sito di iniezione intramuscolare nel topo. I dati ottenuti dimostrano che le tre preparazioni di tossina botulinica presentano una diffusione limitata ai muscoli adiacenti al sito di iniezione e che le tre formulazioni hanno un profilo di diffusione molto simile tra loro.

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Tipo di EPrint:Tesi di dottorato
Relatore:Montecucco, Cesare
Correlatore:Rossetto, Ornella
Dottorato (corsi e scuole):Ciclo 21 > Scuole per il 21simo ciclo > BIOSCIENZE > BIOLOGIA CELLULARE
Data di deposito della tesi:28 Gennaio 2009
Anno di Pubblicazione:31 Gennaio 2009
Parole chiave (italiano / inglese):tossine botuliniche - muscolo scheletrico - N-CAM - atene pesanti della miosina
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/15 Biologia farmaceutica
Area 05 - Scienze biologiche > BIO/19 Microbiologia generale
Area 05 - Scienze biologiche > BIO/09 Fisiologia
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Biomediche Sperimentali
Codice ID:1587
Depositato il:28 Gen 2009
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