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

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