| dc.description.abstract | 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. | |
