Date of Award
Doctor of Philosophy (PhD)
Virginia M. Lee
Cytoplasmic alpha-synuclein (a-syn) aggregates, including Lewy bodies (LBs), are pathological hallmarks of a number of neurodegenerative diseases, most notably Parkinson's disease (PD). Activation of intracellular protein degradation pathways (Pdps) to eliminate these aggregates has been proposed as a therapeutic approach for PD and other synucleinopathies, but the interplay between LB-like a-syn aggregates and Pdps is not completely understood. Here, we investigate this interplay by utilizing a recently developed cellular model in which intracellular LB-like a-syn inclusions accumulate after delivery of pre-formed a-syn fibrils (Pffs) into a-syn-expressing HEK293 cells or cultured primary neurons. This thesis describes the interplay between LB-like aggregates and Pdps, as well as the examination and optimization of the cellular model for this study. We demonstrate that the efficiency of the model can be greatly improved by use of mutant a-syn expressing cells and truncated Pffs. We also show that only a minute amount of Pffs that gain access to cells, and the LB-like aggregates are primarily composed of endogenous a-syn, thereby rendering levels of insoluble endogenous a-syn an excellent read-out for our study. Utilizing the optimized model, we show that a-syn inclusions cannot be effectively degraded by Pdps, even though they colocalize with essential components of these pathways. Furthermore, once formed, a-syn aggregates persist even after reduction of soluble a-syn levels, suggesting that pathological a-syn inclusions are refractory to clearance. Importantly, while proteasome function appears unaltered, we find that a-syn aggregates impair macroautophagy by reducing autophagosome clearance, which may play a role in the increased cell death observed in aggregate-bearing cells. Our results indicate that, after accumulation LB-like of a-syn aggregates, activation of Pdps may not effectively clear pathology or enhance cell survival. However, treatments that could prevent the inhibitory effect of a-syn aggregates on autophagy may be beneficial. We believe this work improves our understanding of the role of a-syn aggregates in synucleinopathies, and it may contribute to the efforts to develop potential therapies for these devastating diseases.
Tanik, Selcuk Aski, "The Interplay Between Lewy Body-Like Alpha-Synuclein Aggregates nd Protein Degradation Pathways in Cell-Based Model of Parkinson's Disease" (2013). Publicly Accessible Penn Dissertations. 705.