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<p dir="ltr">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.</p><p dir="ltr">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.</p><p dir="ltr">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.</p>

Parkinson's Disease (PD) is a neurodegenerative disorder characterized by motor and nonmotor symptoms, including gastrointestinal (GI) dysfunction. Symptoms such as constipation, bloating, and nausea often show early signs before the onset of motor symptoms. Recent research has suggested an essential role of the human gut microbiome and gut dysbiosis, an imbalance in gut microbiota composition, in the pathogenesis of PD. This thesis aims to assess the potential relationship between gut dysbiosis and PD by conducting a meta-analysis, focusing on differences in gut microbiota between PD patients and healthy controls and evaluating their implications for GI dysfunction and PD progression. The primary objectives of this meta-analysis are to (1) compare gut microbiota composition between PD patients and healthy controls via quantitative statistical metrics; (2) identify bacterial taxa differences and their functional roles; (3) assess whether these changes support the hypothesis that gut dysbiosis contributes to α-synuclein (α-SYN) aggregation and PD progression; and (4) evaluate the impact of study methodology and heterogeneity on the reported outcomes. The results revealed no statistically significant differences in alpha diversity between PD patients and controls, likely influenced by substantial heterogeneity across studies. However, specific bacterial taxa were consistently altered, with increased levels of pro-inflammatory bacteria and decreased levels of SCFA-producing bacteria in PD patients. This meta-analysis provides insights into the potential role of gut dysbiosis in PD, suggesting that changes in specific bacterial taxa may contribute to gut inflammation, increased gut permeability, and α-SYN aggregation. The findings highlight the complexity of the gut-brain axis in PD and the need for further longitudinal studies to understand the underlying mechanisms and explore targeted therapeutic intervention.

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