Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Neuroscience

Abstract

The misfolding and subsequent accumulation of alpha-synuclein (α-syn) is central to the pathogenesis of Parkinson's disease (PD). Several lines of evidence suggest pathological α-syn spread cell-to-cell via a "prion-like" mechanism. Furthermore, this pathological α-syn is capable of "seeding" further misfolding of non-pathological α-syn, converting them to the pathological form. While a vast body of both genetic and experimental evidence indicates that α-syn is critical to PD development, how α-syn induces progressive neuronal dysfunction and cell death remains unclear.Autophagy, conventional for macroautophagy, is the primary degradation pathway for α-syn aggregates. Autophagy also influences the unconventional secretion of both pathological and non-pathological α-syn. Evidence ranging from genetic, experimental, and PD brain tissue strongly implicate impaired autophagy as both a symptom and contributor to disease pathology. Additionally, autophagic dysfunction influences the secretion of pathological α-syn via extracellular vesicles (EVs). Notwithstanding, methods to identify and characterize subpopulations of EVs from the total population are lacking. To address this, an imaging-based workflow utilizing immunofluorescence staining and quantitative fluorescent microscopy was formulated to assess the protein composition to characterize individual EVs via Multiplexed Analysis of Co-localization (EV-MAC). Using this EV-MAC workflow secreted α-syn associated EVs were analyzed in the context of PD pathological stimuli. Our lab previously showed that treating cells with oligomerized, α-syn fibrils results in their endocytosis into endo/lysosomal compartments, where they induced rupture, and are then recruited to the autophagic-lysosomal pathway. PD brain tissue stained for the known lysosomal rupture marker, galectin-3 (Gal3), revealed pathological α-syn aggregates were readily Gal3 positive, suggesting a potential link between lysosomal rupture, Gal3, and α-syn accumulation. However, like α-syn, Gal3 is unconventionally secreted in association with EVs and during autophagy impairment. Yet, whether Gal3 or lysosomal rupture affects α-syn secretion, and the underlying mechanisms by which this process could occur are unknown. Here, evidence for a cellular mechanism that explains the cell-to-cell transfer of pathological forms of α-syn is provided. We demonstrate lysosomal rupture, Gal3 recruitment and, in association with its autophagic adaptor protein, tripartite motif containing 16, and autophagy related 16 like 1, stimulate α-syn secretion via an unconventional autophagic pathway. Collectively, this work may contribute to improved diagnostic methods and therapeutics for synucleinopathies.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Share

COinS