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Ursodeoxycholic Acid as a Putative Treatment for Parkinson’s Disease: In vitro study of the cytoprotective effect

Karami, Mohammed (2019) Ursodeoxycholic Acid as a Putative Treatment for Parkinson’s Disease: In vitro study of the cytoprotective effect. PhD thesis, University of Sheffield.

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Abstract

Parkinson’s disease (PD) is an incurable neurodegenerative disease of the elderly. Although the precise molecular mechanism is unknown, accumulative evidence suggests the involvement of mitochondrial dysfunction. Accordingly, a previous drug screening study was carried out in our group with the aim of identifying compounds that rescue mitochondrial function in fibroblasts from PD patients with parkin mutation. Based on the findings in that study, ursodeoxycholic acid (UDCA) was presented as a putative novel treatment for PD. UDCA is an approved treatment for primary biliary cirrhosis. Therefore, its adoption should require less time and cost than de novo drugs. The main aim of this project is to investigate the rescue effect of UDCA in the context of PD. Our group previously described increased activity in each of the individual respiratory chain enzymes following treatment with Ursocholanic acid (UCA), a structurally similar compound. Therefore, we first hypothesised that the rescue effect of UCA/UDCA involves upregulation in mitochondrial biogenesis. However, upon following the treatment protocol used with parkin patient fibroblasts, we were unable to demonstrate an increase in the expression of mitochondrial proteins and mtDNA copy number. Subsequently, we investigated the ability of UDCA to activate the Nrf2 pathway, which is one of the most important antioxidant pathways in cells and has been reported to mediate the action of UDCA in hepatobiliary-related research. In this project we used a specifically designed reporter cell line to assess Nrf2 activation. As a transcriptional factor, activated Nrf2 binds to a distinct sequence (named antioxidant response element (ARE)) in the promotor of its targeted genes, and drives their transcription. The used reporter cells contain several repeats of the ARE sequence linked to green fluorescent protein (GFP) motif. Thus, the measurement of green fluorescence should correlate with the transcription of the ARE sequences. We found that UDCA increased green fluorescence in a dose-dependent manner. However, this increase was not translated into a reduction in oxidative stress in copper-stressed cells. Moreover, UDCA treatment did not rescue these reporter cells when they were stressed with mitochondrial toxins. The reporter cells are not ideal for investigating treatments for PD since they are non-human and non-neuronal in nature. Therefore, the human neuroblastoma-derived SH-SY5Y cell line was subsequently used. UDCA significantly reduced cytotoxicity and increased ATP levels in SH-SY5Y cells stressed with the mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl hydrazine (CCCP). To investigate whether this rescue effect is mediated via the Nrf2 pathway, we quantified the expression of Nrf2 downstream genes via RT-qPCR; however, no positive results were obtained. Subsequent experiments showed that the rescue effect was associated with significant activation of the AKT pathway (a cell survival pathway that has been reported to mediate the action of UDCA). Interestingly, the inhibition of AKT abolished the beneficial effect on ATP but not the cytotoxicity, indicating that the mechanism of action of UDCA is not entirely dependent on AKT. The effect of UDCA treatment on the phosphorylation of selected AKT downstream targets was investigated using western blotting and immunocytochemistry; however, no positive results were obtained. More work is needed to identify AKT/non-AKT downstream targets that are activated/inhibited upon UDCA treatment.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield)
The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Medicine (Sheffield)
Depositing User: Mr. Mohammed Karami
Date Deposited: 01 Apr 2019 09:23
Last Modified: 01 Apr 2019 09:23
URI: http://etheses.whiterose.ac.uk/id/eprint/23511

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