Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive loss of motor neurons. Although many years of research have clarified some of the pathogenetic mechanisms involved in the disease, there is still a need to identify molecular targets for the development of effective therapies. The aim of this thesis was to investigate the involvement of the Axl-Gas6 receptor system in ALS. Axl is a tyrosine kinase receptor belonging to the TAM family and is expressed in the central nervous system (CNS) at the level of neurons, microglia, astrocytes and oligodendrocytes. Gas6 is the endogenous ligand of Axl and is expressed in the CNS by neurons. Among the functions reported in the literature of the Axl-Gas6 axis are: (i) cell survival and/or proliferation mediated by ERK/PI3K cascades or the PI3K/Akt pathway; (ii) elimination of apoptotic cells via induction of phagocytosis by glial cells. Evidence exists for an involvement of Axl-Gas6 axis alterations in neurodegenerative diseases such as Alzheimer's (AD) and multiple sclerosis (MS) or in tumors such as gliomas; however, the role of Axl-Gas6 in ALS has never been described. In this study, by means of immunohistochemistry and in situ hybridization techniques, the localization of the Axl receptor and its ligand Gas6 in the spinal cord was investigated in an animal model of the disease, the transgenic rat expressing human SOD1 mutated to G93A. By using selective antibodies for the transmembrane portion of Axl, or its extracellular domain and phosphorylated receptor, it was possible to reveal changes in the expression levels of the receptor and its ligand in motor neurons, at early stages of the disease, suggesting a possible involvement of Axl-Gas6 axis dysfunction in the mechanisms leading to motor neuron degeneration. These alterations are followed, in the advanced stages of the disease, by increased levels of receptor expression in glial cells, unaccompanied by ligand changes in this cell type, suggesting a possible role of Axl-Gas6 axis alterations also in modulating neuroinflammatory responses during disease progression. Overall, therefore, this study lays the foundation for identifying the Axl-Gas6 pathway as a potential new therapeutic target in ALS.
La sclerosi laterale amiotrofica (SLA) è una malattia neurodegenerativa caratterizzata dalla progressiva perdita dei motoneuroni. Nonostante i molti anni di ricerca abbiano permesso di chiarire alcuni dei meccanismi patogenetici coinvolti nella malattia, vi è ancora necessità di individuare dei target molecolari per lo sviluppo di terapie efficaci. Scopo di questa tesi è stato quello di approfondire il coinvolgimento del sistema recettoriale Axl-Gas6 nella SLA. Axl è un recettore tirosin-chinasico appartenente alla famiglia TAM ed è espresso nel sistema nervoso centrale (SNC) a livello dei neuroni, della microglia, degli astrociti e oligodendrociti. Gas6 è il ligando endogeno di Axl ed è espresso nel SNC dai neuroni. Tra le funzioni riportate in letteratura dell’asse Axl-Gas6 vi sono: i) sopravvivenza e/o proliferazione cellulare mediata dalle cascate ERK/PI3K o dal pathway PI3K/Akt; ii) eliminazione delle cellule apoptotiche tramite induzione della fagocitosi da parte delle cellule gliali. Esistono evidenze di un coinvolgimento di alterazioni dell’asse Axl-Gas6 in malattie neurodegenerative come l’Alzheimer (AD) e la sclerosi multipla (MS) o in tumori come i gliomi; tuttavia, il ruolo di Axl-Gas6 nella SLA non è mai stato descritto. In questo studio, tramite tecniche di immunoistochimica e ibridazione in situ, è stata approfondita la localizzazione del recettore Axl e del suo ligando Gas6 nel midollo spinale in un modello animale della patologia, il ratto transgenico esprimente SOD1 umana mutata in G93A. Tramite l’utilizzo di anticorpi selettivi per il porzione transmembrana di Axl, o del suo dominio extracellulare e del recettore fosforilato è stato possibile mettere in evidenza delle variazioni nei livelli di espressione del recettore e del suo ligando nei motoneuroni, agli stadi precoci della malattia, che suggeriscono un possibile coinvolgimento di disfuzioni dell’asse Axl-Gas6 nei meccanismi che portano a degenerazione motoneuronale. Queste alterazioni sono seguite, negli stadi avanzati della malattia, da un incremento dei livelli di espressione del recettore nelle cellule gliali, non accompagnati da variazioni del ligando in questa tipologia cellulare; il che suggerisce un possibile ruolo di alterazioni dell’asse Axl-Gas6 anche nella modulazione delle risposte neuroinfiammatorie durante la progressione della malattia. Nel complesso, quindi, questo studio getta le basi per individuare nel pathway Axl-Gas6 un potenziale nuovo target terapeutico nella SLA.
Studio del ruolo del recettore Axl e del suo ligando Gas6 in un modello animale di Sclerosi Laterale Amiotrofica Familiare
BANDIRALI, LORIS
2021/2022
Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive loss of motor neurons. Although many years of research have clarified some of the pathogenetic mechanisms involved in the disease, there is still a need to identify molecular targets for the development of effective therapies. The aim of this thesis was to investigate the involvement of the Axl-Gas6 receptor system in ALS. Axl is a tyrosine kinase receptor belonging to the TAM family and is expressed in the central nervous system (CNS) at the level of neurons, microglia, astrocytes and oligodendrocytes. Gas6 is the endogenous ligand of Axl and is expressed in the CNS by neurons. Among the functions reported in the literature of the Axl-Gas6 axis are: (i) cell survival and/or proliferation mediated by ERK/PI3K cascades or the PI3K/Akt pathway; (ii) elimination of apoptotic cells via induction of phagocytosis by glial cells. Evidence exists for an involvement of Axl-Gas6 axis alterations in neurodegenerative diseases such as Alzheimer's (AD) and multiple sclerosis (MS) or in tumors such as gliomas; however, the role of Axl-Gas6 in ALS has never been described. In this study, by means of immunohistochemistry and in situ hybridization techniques, the localization of the Axl receptor and its ligand Gas6 in the spinal cord was investigated in an animal model of the disease, the transgenic rat expressing human SOD1 mutated to G93A. By using selective antibodies for the transmembrane portion of Axl, or its extracellular domain and phosphorylated receptor, it was possible to reveal changes in the expression levels of the receptor and its ligand in motor neurons, at early stages of the disease, suggesting a possible involvement of Axl-Gas6 axis dysfunction in the mechanisms leading to motor neuron degeneration. These alterations are followed, in the advanced stages of the disease, by increased levels of receptor expression in glial cells, unaccompanied by ligand changes in this cell type, suggesting a possible role of Axl-Gas6 axis alterations also in modulating neuroinflammatory responses during disease progression. Overall, therefore, this study lays the foundation for identifying the Axl-Gas6 pathway as a potential new therapeutic target in ALS.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.
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https://hdl.handle.net/20.500.14239/14812