Exploration of 20s Proteasome Stimulation as a Therapeutic Approach to Parkinson's Disease

Rachel Anne Coleman, Purdue University

Abstract

Parkinson's disease (PD) is a detrimental ncurodegenerative disorder characterized by the presence of large protein aggregates in the brain called Lewy bodies, which are primarily composed of the protein a-synuclein (aSyn). Due to the dysregulation of aSyn levels in PD, controlling its levels through the manipulation of protein degradation pathways has been suggested as a therapeutic avenue for the treatment of PD and related diseases. Although aSyn is known to be degraded through the autophagy and proteasome pathways, it is one of only a few known substrates of the ubiquitin-independent proteasome pathway, which utilizes the 20S core particle of the proteasome (20S CP) to degrade proteins. We therefore hypothesize that small molecule stimulation of the 20S CP will enhance aSyn degradation and reduce aSyn pathology, providing a therapeutic benefit in PD models. We began our studies by developing a fluorescence resonance energy transfer (FRET) reporter assay to monitor 20S CP activity and screen for small molecule stimulators. This assay provides a greater dynamic range to detect 20S CP stimulation compared to the most commonly used assay to monitor proteasome activity. Using the FRET assay, we were able to identify a number of novel 20S CP stimulators that differ in structure as well as potency and degree of stimulation. We next evaluated the ability of four small molecule stimulators to enhance protein degradation by the 20S CP in a biochemical assay using 15 different purified proteins. These 15 proteins include known substrates of the 20S CP and vary in size and degree of disorder. From this assay, we demonstrate that a 20S CP stimulator is likely to enhance the degradation of highly disordered proteins, such as aSyn, but the effect on other protein levels appears to be distinct for each stimulator. Two of our more potent stimulators, AM-404 and miconazole, were used with the proteasome inhibitor bortezomib for subsequent studies in HEK-293T cells in which we corroborated the results of our biochemical assay. While both AM-404 and miconazole were shown to impact highly disordered proteins, there was not much overlap between the proteins shown to be affected by each stimulator. Due to the distinct effect of each stimulator on protein regulation by the 20S CP, this study indicates the potential of tailoring a small molecule 20S CP stimulator to enhance the degradation of particular substrates. Since AM-404 and miconazole were shown to impact 20S CP activity in different ways, we next evaluated whether either stimulator would be able to prevent the aSyn-induced inhibition of the 20S CP. High levels of a@Syn have been shown to lead to proteasome impairment in biochemical and cell studies. We confirm 20S CP impairment in the presence of micromolar amounts of a Syn, and we demonstrate that miconazole, but not AM-404, is effective at maintaining 20S CP activity in the presence of increasing concentrations of aSyn. We also show that a Syn-overexpressing PC12 cells (PC12 C4 cells) display reduced proteasome activity compared to the parent cell line. Miconazole and AM-404 increased proteasome activity in PC 12 C4 cells, which were more sensitive to 20S CP stimulation than non-transfected PC 12 cells, but miconazole was shown to be more effective at modulating aSyn phosphorylated at Ser129 in PC 12 C4 cells.

Degree

Ph.D.

Advisors

Trader, Purdue University.

Subject Area

Pathology|Biochemistry|Cellular biology|Neurosciences

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