The Huntington gene in its wild-type version controls the development of the central nervous system, particularly the formation of the neural tube (Lo Sardo et al., Nature Neuroscience 2012). By using mouse embryonic stem (mES) cells subjected to a special differentiation protocol able to reproduce in vitro the development of the neural tube (Ying et al., 2003), we showed that huntingtin plays a critical role in neurulation process (Lo Sardo et al., 2012). This differentiation protocol allows the formation after 7 days in vitro of three-dimensional structures called neural rosettes, showing many of the characteristics of the neural tube observed in vivo. Huntingtin (HTT) is indispensable for the interactions between neuroepithelial cells that constitute the neural rosettes. In fact, mES cells homozygous knock-out for the Huntington gene undergo neural differentiation but do not form neural rosettes (rosetteless phenotype) (Lo Sardo et al., Nature Neuroscience 2012). The purpose of this thesis work was to identify which of the Huntington gene sequences play a significant role in rosettes formation. We focused on the fragment of the gene encoding for the N-terminal since previous studies have shown that it is essential for neurulation in vitro. More specifically, we analyzed the PolyQ stretch located in the N-terminal region of the protein as it elongates in deuterostome evolution with the increase in complexity of the central nervous system in this branch. Therefore during my thesis project mES cells homozygous knock-out for the Huntington gene were transfected with a construct expressing the first 548 amino acids of HTT lacking the polyQ tract or with the polyQ interrupted by two amino acidic substitutions with an alanine and a proline. The obtained cell lines were subjected to a neural differentiation protocol to test whether removal of the polyQ tract from HTT and/or its interruption could significantly reduce the ability of the protein to promote neural rosettes formation.
Il gene Huntington nella sua versione wild-type controlla lo sviluppo del sistema nervoso centrale, in particolare la formazione del tubo neurale (Lo Sardo et al., Nature Neuroscience 2012). Precedenti studi condotti nel laboratorio in cui ho svolto la mia tesi mostrano che le cellule staminali embrionali murine (mES) sottoposte a un particolare protocollo di differenziamento in grado di ripercorrere in vitro lo sviluppo del tubo neurale (Ying et al., 2003), formano dopo 7 giorni strutture tridimensionali chiamate rosette neurali. Le rosette possiedono numerose caratteristiche proprie del tubo neurale osservate in vivo. I nostri studi hanno evidenziato che l'Huntingtina è indispensabile proprio per le interazioni tra cellule neuroepiteliali che costituiscono le rosette neurali durante la neurulazione in vitro (Lo Sardo et al., Nature Neuroscience 2012). Infatti, cellule mES omozigoti knock-out per il gene Huntington sottoposte a differenziamento neurale generano precursori neuroepiteliali non organizzati in rosette (fenotipo rosetteless). Lo scopo di questa tesi è identificare quali sequenze o porzioni del gene Huntington rivestono un ruolo rilevante nella neurulazione in vitro. Abbiamo posto l’attenzione sulla porzione del gene che codifica per il tratto N-terminale della proteina Huntingtina (HTT) poiché studi precedenti hanno evidenziato che i primi 548 amminoacidi della HTT sono essenziali per il processo di neurulazione in vitro. Più nello specifico, abbiamo analizzato il tratto PoliQ dell’HTT localizzato proprio nella regione N-terminale della proteina in quanto sembra allungarsi in concomitanza dell’aumento di complessità del Sistema Nervoso Centrale durante l’evoluzione dei deuterostomi. Pertanto, durante il mio progetto di tesi, le cellule mES omozigoti knock-out per il gene Huntington sono state trasfettate con un costrutto esprimente il frammento di huntingtina costituto dai primi 548 aminoacidi privi del tratto poliQ o con un poliQ interrotto dalla presenza di alanine o proline. Le linee cellulari ottenute sono state quindi sottoposte al protocollo di differenziamento neurale per valutare se la rimozione del tratto poliQ dell’HTT o la sua interruzione potesse inficiare la formazione delle rosette neurali.
Ruolo del tratto PoliQ nella proteina Huntingtina durante la neurogenesi.
CAFFARELLI, GIULIA
2015/2016
Abstract
The Huntington gene in its wild-type version controls the development of the central nervous system, particularly the formation of the neural tube (Lo Sardo et al., Nature Neuroscience 2012). By using mouse embryonic stem (mES) cells subjected to a special differentiation protocol able to reproduce in vitro the development of the neural tube (Ying et al., 2003), we showed that huntingtin plays a critical role in neurulation process (Lo Sardo et al., 2012). This differentiation protocol allows the formation after 7 days in vitro of three-dimensional structures called neural rosettes, showing many of the characteristics of the neural tube observed in vivo. Huntingtin (HTT) is indispensable for the interactions between neuroepithelial cells that constitute the neural rosettes. In fact, mES cells homozygous knock-out for the Huntington gene undergo neural differentiation but do not form neural rosettes (rosetteless phenotype) (Lo Sardo et al., Nature Neuroscience 2012). The purpose of this thesis work was to identify which of the Huntington gene sequences play a significant role in rosettes formation. We focused on the fragment of the gene encoding for the N-terminal since previous studies have shown that it is essential for neurulation in vitro. More specifically, we analyzed the PolyQ stretch located in the N-terminal region of the protein as it elongates in deuterostome evolution with the increase in complexity of the central nervous system in this branch. Therefore during my thesis project mES cells homozygous knock-out for the Huntington gene were transfected with a construct expressing the first 548 amino acids of HTT lacking the polyQ tract or with the polyQ interrupted by two amino acidic substitutions with an alanine and a proline. The obtained cell lines were subjected to a neural differentiation protocol to test whether removal of the polyQ tract from HTT and/or its interruption could significantly reduce the ability of the protein to promote neural rosettes formation.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/21644