In this work, I applied advanced microscopy techniques to study the molecular mechanisms of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease. The toxicity of α-syn is likely related to its interaction with the neuronal membrane. It is known that α-syn is present in both the cytosolic and extracellular space, and it has been demonstrated that both components have a role in neurodegenerative processes. To study the effect α-syn on lipid membranes, I used two complex lipid mixtures, preparing lipid bilayers that modeled the external and internal leaflets of the neuronal membrane. Exploiting this original approach, I investigated how α-syn may act on membrane structure and stability when it presents in the cytosol or the extracellular space. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM). I used atomic force microscopy (AFM) in different acquisition modes, and I complemented the results with QCM-D, and vesicle leakage experiments. In a second work, I characterized amyloid aggregates formed by α-syn, applying a correlative technique based on the coupling between AFM and stimulated emission depletion (STED) microscopy. In particular, I focused on the definition of the eventual influence that the presence of fluorescent dye molecules can have on the aggregation process of the peptide. I demonstrated that the use of a site-specific labeling method is fundamental to avoid modification of the aggregation process.

Advanced microscopy techniques applied to in-vitro study of Alpha synuclein aggregation and toxicity mechanism

JADAVI, SAMIRA
2022

Abstract

In this work, I applied advanced microscopy techniques to study the molecular mechanisms of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease. The toxicity of α-syn is likely related to its interaction with the neuronal membrane. It is known that α-syn is present in both the cytosolic and extracellular space, and it has been demonstrated that both components have a role in neurodegenerative processes. To study the effect α-syn on lipid membranes, I used two complex lipid mixtures, preparing lipid bilayers that modeled the external and internal leaflets of the neuronal membrane. Exploiting this original approach, I investigated how α-syn may act on membrane structure and stability when it presents in the cytosol or the extracellular space. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM). I used atomic force microscopy (AFM) in different acquisition modes, and I complemented the results with QCM-D, and vesicle leakage experiments. In a second work, I characterized amyloid aggregates formed by α-syn, applying a correlative technique based on the coupling between AFM and stimulated emission depletion (STED) microscopy. In particular, I focused on the definition of the eventual influence that the presence of fluorescent dye molecules can have on the aggregation process of the peptide. I demonstrated that the use of a site-specific labeling method is fundamental to avoid modification of the aggregation process.
File in questo prodotto:
File Dimensione Formato  
phdunige_4619376.pdf

embargo fino al 04/05/2023

Descrizione: description of experimental results
Tipologia: Tesi di dottorato
Dimensione 5.13 MB
Formato Adobe PDF
5.13 MB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/1080907
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact