INVESTIGATING PROTEIN AGGREGATION IN NEURODEGENERATIVE DISEASES USING FLUORESCENCE LIFETIME IMAGING MICROSCOPY

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.

A time-gated fluorescence lifetime imaging microscope system was developed to monitor the evolution of seeded aggregation in primary neurons in the context of endocytic processes that have yet to be well explored. This aggregation monitoring was enabled by measuring self-quenching-induced fluorescence lifetime changes of alpha-synuclein-fluorophore fusion proteins, providing a sensitive aggregate detection method. Results from this work demonstrate that both escape and retention of fibrillar seeds from endocytic compartments are seeding pathways for aggregation. In addition, a novel imaging scheme was developed using fluorescence lifetime measurements of tethered Förster resonance energy transfer (FRET) reporters to probe membrane-induced alpha-synuclein aggregation. Using this method in neurons enabled deciphering of which intracellular membrane surfaces likely play a role in alpha-synuclein aggregation.

This work used fluorescence lifetime imaging to enable insights into the underlying mechanisms of alpha-synuclein aggregation in neurons. This has broader applications to other prion-like neurodegenerative diseases. These insights further our understanding of neurodegenerative disease etiology and can inform more effective treatments. Additionally, an approach to noise estimation that enables accurate extraction of fluorescence lifetime information in the presence of substantial detector noise is presented. This will enable longer-term multi-time-point fluorescence lifetime imaging of aggregation in neurons.