Parkinson’s Disease (PD) is a neurodegenerative pathology characterized by a chronic and progressive loss of dopaminergic neurons mostly located in the Substantia Nigra pars compacta (SNc), with clear histopathological hallmarks related to large α-synuclein aggregates called Lewy bodies (LB). Like other neurodegenerative diseases, PD shows sporadic and familiar forms, and among these last ones the GBA1 (glucocerebrosidase) mutations represents one of the main risk factors since they are involved in the dysfunction of the right degradative lysosomal activity, which is closely related to the autophagic process. In this context Ambroxol – a mucolytic drug used to treat respiratory conditions – has gained interest as a potential lysosomal chaperone and it’s getting tested in several clinical trials with the aim of increasing the pool of pharmacological interventions at our disposal to treat the disease. In this study, the SH-SY5Y human neuroblastoma cellular line – at first undifferentiated and then differentiated toward a neuronal phenotype through retinoic acid – was used as an in vitro model to study the effects of Ambroxol in basal conditions and in presence of autophagy inhibition (obtained through Bafilomycin). α-synuclein levels were evaluated both intra- and extra-cellularly, together with different autophagic, lysosomal and exocytic markers to verify how these processes are influenced by the drug. These effects were studied after treating the cells with Ambroxol 60µM for three days, evaluating then the protein expression through Western Blot; for what concerns exocytosis, the Dot Blot methodology was used instead. Regarding the PBMC obtained from a subgroup of patients involved in a clinical trial with Ambroxol, the levels of gene and protein expression of different autophagy markers were evaluated through RT-qPCR and Western Blot. The results obtained suggest that Ambroxol can influence autophagy by increasing protein levels of p62 and LC3-II, and of CD63 for exocytosis. The PBMC analysis instead didn’t highlight any appreciable effect of Ambroxol on the autophagy markers, except for LAMP2A, a lysosomal receptor related to the chaperone-mediated autophagy (CMA), whose protein levels show the tendency to increase in drug-treated patients, while the gene expression is reduced. On the other hand, Bafilomycin, which was administered on the second day for 18h, has potentiated some of the effects observed with Ambroxol alone, although it was irrelevant about p62 exocytosis. This could depend on a bafilomycin-induced autophagic vesicles degradation block which, in combination with an Ambroxol-dependent increase in these vesicles production, might lead to an augmentation of the levels of the proteins there localized. An alternative is the possibility that Ambroxol might behave as an autophagy inhibitor, enhancing the results obtained through Bafilomycin thus working similarly to this agent. Overall, these results show that Ambroxol can increase exocytic and autophagic markers, although the nature of this phenomenon is still to be clarified. Moreover, it’s been observed that mononuclear cells can’t properly reflect these changes at a peripheral level, making further studies necessary to better understand the molecular mechanisms underlying the neuroprotective effect of Ambroxol.
La malattia di Parkinson (PD) è una patologia neurodegenerativa caratterizzata dalla perdita progressiva e cronica di neuroni dopaminergici principalmente a livello della Substantia Nigra pars compacta (SNc), con evidenti tratti istopatologici legati alla presenza di accumuli di α-sinucleina chiamati corpi di Lewy (LB). Come diverse malattie neurodegenerative, anche il PD presenta forme sporadiche e familiari, dove tra queste ultime uno dei principali fattori di rischio è rappresentato dalle mutazioni del gene GBA1 (glucocerebrosidasi), coinvolte nella disfunzione dell’attività degradativa lisosomiale che risulta strettamente legata al processo autofagico. In questo contesto ha ottenuto risalto Ambroxolo – un farmaco mucolitico usato per il trattamento di malattie respiratorie – come potenziale chaperone lisosomiale che sta venendo attualmente testato in diversi trial clinici con la prospettiva di ampliare le terapie farmacologiche a disposizione contro la malattia. In questo studio è stata utilizzata una linea cellulare di neuroblastoma umano SH-SY5Y, indifferenziata e dopo differenziamento verso un fenotipo neuronale tramite acido retinoico, come modello in vitro per studiare l’effetto di Ambroxolo in condizioni basali e in presenza di inibizione dell’autofagia (ottenuta tramite Bafilomicina). Sono stati valutati i livelli di α-sinucleina intra- ed extra-cellulari e di diversi marker autofagici, lisosomiali e di esocitosi, per verificare come questi processi vengano influenzati dal farmaco. Tali effetti sono stati studiati trattando le cellule con Ambroxolo 60µM per tre giorni, andando poi a valutare l’espressione di diverse proteine mediante Western Blot; per quanto riguarda l’esocitosi invece, si è adottata la metodica del Dot Blot. Nei PBMC ottenuti da un sottogruppo di pazienti partecipanti ad un clinical trial su Ambroxolo sono stati valutati i livelli di espressione genica e proteica di diversi markers autofagici mediante RT-qPCR e Western Blot. I risultati ottenuti suggeriscono che Ambroxolo sia in grado di influenzare l’autofagia aumentando i livelli proteici di p62 e LC3-II, oltre che di CD63 per quanto riguarda l’esocitosi. Le analisi condotte sui PBMC non hanno evidenziato un apprezzabile effetto di Ambroxolo sui marker autofagici, ad eccezione di LAMP2A, recettore lisosomiale della chaperone-mediated autophagy (CMA), i cui livelli proteici mostrano una tendenza all’aumento, accompagnata da una ridotta espressione genica, nei pazienti trattati con il farmaco. L’applicazione di Bafilomicina, somministrata al secondo giorno per 18h, ha inoltre incrementato alcuni degli effetti osservati con il solo Ambroxolo, sebbene nell’esocitosi di p62 sia risultata ininfluente. Questo potrebbe dipendere da un blocco della degradazione delle vescicole autofagiche indotto dalla stessa che, in sinergia con un aumento della produzione di queste vescicole mediato da Ambroxolo, potrebbe portare a un complessivo aumento dei livelli di proteine ivi localizzate. Un’alternativa è la possibilità che anche Ambroxolo si comporti come un inibitore autofagico, aumentando i risultati ottenuti con Bafilomicina e lavorando similmente a essa. Complessivamente questi risultati indicano che Ambroxolo è in grado di aumentare i livelli di esocitosi e di marker autofagici, anche se la natura di questo incremento rimane ancora da chiarire. Inoltre si è mostrato come le cellule mononucleate non siano in grado di riflettere adeguatamente i cambi di questi fenomeni a livello periferico, rendendo pertanto necessari ulteriori studi per chiarire i meccanismi molecolari dell’effetto neuroprotettivo di Ambroxolo.
EFFETTI DI AMBROXOLO SU AUTOFAGIA ED ESOCITOSI IN UN MODELLO IN VITRO DI MALATTIA DI PARKINSON
FONTANA, LUCA
2024/2025
Abstract
Parkinson’s Disease (PD) is a neurodegenerative pathology characterized by a chronic and progressive loss of dopaminergic neurons mostly located in the Substantia Nigra pars compacta (SNc), with clear histopathological hallmarks related to large α-synuclein aggregates called Lewy bodies (LB). Like other neurodegenerative diseases, PD shows sporadic and familiar forms, and among these last ones the GBA1 (glucocerebrosidase) mutations represents one of the main risk factors since they are involved in the dysfunction of the right degradative lysosomal activity, which is closely related to the autophagic process. In this context Ambroxol – a mucolytic drug used to treat respiratory conditions – has gained interest as a potential lysosomal chaperone and it’s getting tested in several clinical trials with the aim of increasing the pool of pharmacological interventions at our disposal to treat the disease. In this study, the SH-SY5Y human neuroblastoma cellular line – at first undifferentiated and then differentiated toward a neuronal phenotype through retinoic acid – was used as an in vitro model to study the effects of Ambroxol in basal conditions and in presence of autophagy inhibition (obtained through Bafilomycin). α-synuclein levels were evaluated both intra- and extra-cellularly, together with different autophagic, lysosomal and exocytic markers to verify how these processes are influenced by the drug. These effects were studied after treating the cells with Ambroxol 60µM for three days, evaluating then the protein expression through Western Blot; for what concerns exocytosis, the Dot Blot methodology was used instead. Regarding the PBMC obtained from a subgroup of patients involved in a clinical trial with Ambroxol, the levels of gene and protein expression of different autophagy markers were evaluated through RT-qPCR and Western Blot. The results obtained suggest that Ambroxol can influence autophagy by increasing protein levels of p62 and LC3-II, and of CD63 for exocytosis. The PBMC analysis instead didn’t highlight any appreciable effect of Ambroxol on the autophagy markers, except for LAMP2A, a lysosomal receptor related to the chaperone-mediated autophagy (CMA), whose protein levels show the tendency to increase in drug-treated patients, while the gene expression is reduced. On the other hand, Bafilomycin, which was administered on the second day for 18h, has potentiated some of the effects observed with Ambroxol alone, although it was irrelevant about p62 exocytosis. This could depend on a bafilomycin-induced autophagic vesicles degradation block which, in combination with an Ambroxol-dependent increase in these vesicles production, might lead to an augmentation of the levels of the proteins there localized. An alternative is the possibility that Ambroxol might behave as an autophagy inhibitor, enhancing the results obtained through Bafilomycin thus working similarly to this agent. Overall, these results show that Ambroxol can increase exocytic and autophagic markers, although the nature of this phenomenon is still to be clarified. Moreover, it’s been observed that mononuclear cells can’t properly reflect these changes at a peripheral level, making further studies necessary to better understand the molecular mechanisms underlying the neuroprotective effect of Ambroxol.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/31633