Optimization of mouse brain-derived exosome isolation method and a study of exosomal roles in the pathological propagation of alpha-synuclein aggregates in Parkinson’s disease

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Perustieteiden korkeakoulu | Master's thesis
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Date

2020-06-15

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Major/Subject

Human Neuroscience and Technology

Mcode

SCI3061

Degree programme

Master’s Programme in Life Science Technologies

Language

en

Pages

6+59

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Abstract

Exosomes are small extracellular vesicles that are secreted by all cells and have diverse functions depending on their cellular origin. In the nervous system, exosomes are known to be involved in intercellular communications, carrying molecular cargo and regulation of various physiological processes. In recent decades, exosomes have attracted immense attention due to their implications in many neurodegenerative diseases like Parkinson’s disease (PD) and Alzheimer’s disease. Specifically, mounting evidence suggests that exosomes may be key mediators of the “prion-like” propagation in PD pathology as they are known to secrete alpha synucleins into the extracellular space and transfer them between the neighboring cells. As the second most prevalent neurodegenerative disease, PD is notorious for the progressive loss of dopaminergic neurons in the substantia nigra pars compacta that severely affect the patients’ quality of life with no disease modifying therapies available to this date. Thus, the possibilities of uncovering the underlying mechanisms and developing potential therapeutics or biomarkers for PD using exosomes are positively appealing. However, the lack of standardized protocols and ambiguous definitions has been raising concerns regarding the advancement of exosome research despite the enormous potential these nano-sized vesicles may harbor. In this thesis, three commonly used exosome isolation methods with varying conditions were investigated to establish an optimized protocol that would be efficient and practical for isolating exosomes from mouse brain tissues. The results demonstrated that isolating exosomes from frozen brain tissues using the sucrose density gradient centrifugation method was optimal considering the ease of performance and the high purity of resulting exosomes. Moreover, the importance of adequately dissociating the brain tissues using an effective enzyme like collagenase type III to minimize disruption of the cells prior to the isolation process has been reaffirmed. In addition, a preliminary study of exosomal roles in the pathological propagation of alpha synuclein aggregates was conducted using the isolated exosomes from the mice injected with alpha synuclein preformed fibrils (PFF) to induce PD pathology. An increased concentration of post-translationally modified forms of alpha synuclein were found in the exosomes from the PFF-injected mice although no apparent increase of the total concentration of exosome was observed. Taken together, the modified forms of exosomal alpha synuclein may be substrates of cellular homeostatic processes that occur in the brain to mitigate the disease effect and may represent an intercellular snapshot of the spreading of Parkinson’s pathology at an early stage.

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Supervisor

Carlson, Synnöve

Thesis advisor

Panhelainen, Anne

Keywords

exosome, extracellular vesicles, Parkinson's disease, alpha-synuclein, neurodegenerative disorders, propagation

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https://urn.fi/URN:NBN:fi:aalto-202006213965

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[dipl] Perustieteiden korkeakoulu / SCI