Browsing by Author "Bekker, Minke"

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    An investigation of the neuroprotective properties of curcumin by monitoring autophagy and apoptosis
    (Stellenbosch : Stellenbosch University, 2021-03) Bekker, Minke; Bardien, Soraya; Loos, Ben; Abrahams, Shameemah; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative movement disorder, with a rapidly increasing prevalence and incidence throughout the global population. As current PD therapies only rely on symptomatic treatments, there is an urgent need for the development of neuroprotective therapies, to slow or halt progressive neuronal loss. This strategy, however, is dependent on a better understanding of the pathobiology and pathways underlying PD. Multiple causative factors have been postulated to be involved in the pathobiology of PD, with defective autophagy and the subsequent upregulation of apoptotic cascades hypothesised as main contributing factors. Although the crosstalk between these pathways has become evident, the exact role of autophagy and apoptosis regarding the neuronal fate in PD remains controversial. Curcumin is a polyphenolic plant compound that has been observed to mediate autophagy and apoptosis. Consequently, the aim of the present study was to investigate the potential of curcumin as a PD therapy, and its effect on autophagy and apoptosis in a PD model. Study objectives were set out to achieve this aim, with the first objective being to better understand the interplay between autophagy and apoptosis, by performing a literature review to identify molecular components involved in both pathways. The second objective was to establish an appropriate PD model for the experimental part of the study. The cellular model selected was treatment with a neurotoxin, paraquat, in a commercially available SH-SY5Y neuroblastoma cell line. This objective was executed through performing 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2H-tetrazolium bromide and CyQUANT assays to determine appropriate concentrations for curcumin and paraquat treatment. The third objective was to assess the effect of curcumin in the autophagic pathway, through performing western blotting to measure levels of the autophagic LC3-II, p62 and LAMP2 proteins. Lastly, the fourth objective was to assess the effect of curcumin in the apoptotic pathway in an autophagy-inhibited model. This was executed through performing western blotting to measure the levels of the autophagic Beclin-1 and apoptotic Bax proteins. The literature review pinpointed Bcl-2, JNK, p38 and Akt as proteins with the potential to mediate the activation and/or inhibition of both pathways. It was concluded that novel PD therapies could target these connecting molecular components to mediate the balance between autophagy and apoptosis. Using our PD cellular model, our findings revealed a trend for curcumin treatment to increase the overall abundance of LC3-II protein levels, while also sustaining the protein levels of LAMP2 in the presence of the autophagic inhibitor bafilomycin A1. Curcumin treatment also facilitated the efficient clearance of p62 protein, in comparison to a blunted clearance of p62 protein observed upon paraquat treatment. These findings reflect the potential of curcumin to induce autophagy while also maintaining the balance of autophagic flux. Additionally, a trend for curcumin to increase Beclin- 1 and decrease Bax protein levels in the presence of the autophagic inhibitor, 3-methyladenine, was observed. Curcumin therefore exhibited the potential to counteract autophagic inhibition while also demonstrating anti-apoptotic properties, independently from autophagy. Considering these results and published evidence of curcumin-mediated changes in the expression of Bcl-2, JNK and, Akt, it is suggested that curcumin treatment exhibits the potential to facilitate the interplay between autophagy and apoptosis. Study limitations include the challenge of interpreting autophagic flux through western blotting and the use of an undifferentiated cell line and can be addressed in future work. The findings in this study are of importance, as they may contibute to a better understanding of the pathobiology of PD which could advance the development of novel therapies, to potentially ameliorate the detrimental nature of this disorder.