Investigation of the Small Ubiquitin-like Modifier Pathway in Parkinson's Disease as a Therapeutic Target and Potential Biomarker

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Pountney, Dean

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Munn, Alan

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Advancing age is the biggest risk factor in developing age-related disorders such as cardiovascular disease, cancer, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and other neurodegenerative diseases, regardless of efforts to improve life expectancy. Neurodegenerative diseases comprise of chronic progressive disease conditions that are characterised by the gradual failure of cellular systems leading to neuronal loss. The main hallmark of these diseases is the abnormal deposition of toxic protein aggregates that spread to various brain regions leading to debilitating clinical symptoms affecting a person’s quality of life. Regardless of existing treatments to improve these symptoms, more research is required towards unravelling the mysteries that contribute to the progression of these disorders. Parkinson’s disease (PD), one of the most common neurodegenerative disorder, reflects a progressive loss of dopaminergic neurons in the substantia nigra leading to a wide range of motor deficits. Neuropathological hallmarks of PD comprise of amorphous aggregates that lead to the formation of Lewy bodies containing mainly the misfolded protein component, asynuclein (a-syn), and other substrates such as the small ubiquitin-like modifier-1 (SUMO-1). This thesis investigates the SUMO pathway and its components as a potential therapeutic target and potential biomarker for PD. The two aims of this thesis were to determine the influence of SUMO inhibitors on a cellular model of PD (Chapter 5) and to establish the differential activity of the SUMO pathway in patient-derived PD cell lines compared to age-matched normal control and in response to proteolytic stress (Chapter 6). The experimental work presented in Chapter 5 investigated the inhibition of the SUMO- 1 E1 enzyme of the SUMO pathway using 2 chemical inhibitors, ginkgolic acid (GA) and anacardic acid (AA) in KCl-depolarised SHSY5Y neuroblastoma cells and primary rat neurons. Potassium chloride (KCl) depolarisation has been reported previously to induce aggregate formation and SUMO-1 marked a subset of lysosomes within cytoplasmic inclusions. Immunofluorescence and cell counting were employed to determine the proportion of SUMO-1 positive lysosomes, the frequency of autophagosomes and a-synpositive puncta in KCl depolarised SHSY5Y cells under various concentrations of SUMO inhibitor treatments. Depolarised rat cortical neurons were also analysed to determine the frequency of a-syn-positive aggregates under SUMO inhibition. Western blot analysis of cell lysates subjected to KCl depolarisation was performed to determine the total band integrals of SUMO-1 conjugation, the difference in intensity between SUMO-1 90kDa band and the Hsp90 band as well as the intensity of macroautophagic marker, LC3b, under various concentrations of SUMO inhibitor treatments. Previous studies have suggested that the SUMO pathway is linked to chaperone-mediated autophagy. The results revealed that the SUMOylation inhibitors induced upregulation of macroautopahagy and promoted a-syn aggregate clearance in aggregate-bearing cells. The experimental work presented in Chapter 6 investigated the various SUMO pathway components in patient-derived PD cell lines in comparison to age-matched normal control cell lines and in response to proteasome inhibition. Immunofluorescence and cell counting were employed to determine the proportion of SUMO-1 positive lysosomes and the frequency of protein aggregates in PD cell lines compared to normal controls with and without MG132 treatments. Western blot analysis was employed to determine the levels of SUMO-1 conjugates, levels of Hsp90 and levels of the deSUMOylase, SENP3. Findings in this current chapter identified significant differences in the SUMO components in disease-specific cell lines compared to age-matched controls indicating the potential use as biomarkers for the pre-symptomatic diagnosis of PD.

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Thesis (PhD Doctorate)

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Doctor of Philosophy (PhD)


School of Medical Science

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Parkinson's disease

Therapeutic target



PD cell lines

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