IBSC-BME PhD Defense Announcement for Paula Ivey (Tamara Kinzer-Ursem and Kevin Webb, co-advisors) Everyone is invited to attend the public presentation beginning at 10:30 am. Title: Investigating Protein Aggregation in Neurodegenerative Diseases Using Fluorescence Lifetime Imaging Microscopy Date: July 9th Time: 10:30 am Location: BHEE 317 Committee members: Prof. Kevin Webb (Co-chair), Prof. Tamara Kinzer-Ursem (Co-chair), Prof. Jean-Christophe Rochet, Prof. Krishna Jayant Abstract: Alpha-synuclein protein aggregation, involving the recruitment of native monomeric protein by fibrillar seeds, has been proposed as the event that precipitates Parkinson's disease pathology. However, the specific molecular processes underlying this aggregation are not fully understood, mirroring the limitations seen in understanding the etiology of other prion-like neurodegenerative diseases. There are proposed mechanisms connecting alpha-synuclein aggregation to endocytic processes involving the escape and retention of fibrillar seeds. Additionally, intracellular protein-membrane interactions may also play a role. However, effective methods to probe the evolution of aggregation states with sufficient sensitivity in the context of these cellular processes are lacking. We used a custom-built fluorescence lifetime imaging microscope to monitor the evolution of seeded aggregation in primary neurons in the context of endocytic processes that have not been well explored. This was enabled by measuring self-quenching-induced fluorescence lifetime changes of alpha-synuclein-fluorophore fusion proteins, providing a sensitive aggregate detection method. Our results indicate that both escape and retention of fibrillar seeds from endocytic compartments are seeding pathways for aggregation. In addition, we developed a novel imaging scheme using fluorescence lifetime measurements of tethered Förster resonance energy transfer (FRET) reporters to probe membrane-induced alpha-synuclein aggregation. Employing this method in neurons enabled us to decipher which intracellular membrane surfaces likely play a role in alpha-synuclein aggregation. Fluorescence lifetime imaging enabled insights into the underlying mechanisms of alpha-synuclein aggregation, which has broader applications to other prion-like neurodegenerative diseases. These insights further understanding of neurodegenerative disease etiology and can inform more effective treatments.