Masters Degrees (Molecular Biology and Human Genetics)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/3555

Browse

Browsing Masters Degrees (Molecular Biology and Human Genetics) by browse.metadata.advisor "Abrahams, Shameemah"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Evaluating the neuroprotective effect of curcumin on a PINK1 cell model of Parkinson's disease
    (Stellebosch : Stellenbosch University, 2022-04) Chetty, Devina; Bardien, Soraya; Kenyon, Colin; Abrahams, Shameemah; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a loss of neurons producing the neurotransmitter dopamine. Notably, despite extensive studies that have revealed numerous dysregulated processes associated with PD, fundamental gaps still exist in our knowledge of the disease pathophysiology. Our understanding of the disease includes processes such as mitochondrial dysfunction causing increased oxidative stress and energy failure, as well as misfolded protein accumulation in large inclusions. However, the spatiotemporal sequence of events leading to PD and, importantly, the initial factors that trigger disease onset remain elusive. This challenges the development of effective therapeutic strategies to prevent and cure PD. Moreover, the various side effects associated with conventional pharmaceuticals currently used to treat this disease motivate the search for a natural treatment that can avoid augmenting the suffering associated with an already debilitating disease. It is evident that mitochondrial dysfunction and oxidative stress are likely to be involved in the disease pathogenesis, therefore studying these phenomena in PD may lead to the development of more effective therapeutic strategies. Curcumin is a plant-based polyphenol that has been observed to have antioxidant properties, increase cell viability, and enhance mitochondrial function. Consequently, the aim of the present study was to create a PD cellular model and evaluate the potential protective effects of curcumin. Therefore, we sought to establish an appropriate PD model by transfecting SH-SY5Y cells with wild-type (WT) or G309D mutant PINK1 cDNA, of which the latter has been shown to upregulate dopamine and lead to cytotoxicity. Additionally, since several neurotoxins have been shown to trigger PD, the toxic herbicide paraquat was administered to complement the model with cellular damage and mitochondrial dysfunction. To confirm the model, we performed RT-qPCR to measure gene expression levels of PINK1 and tyrosine hydroxylase (TH), an enzyme in the dopamine synthesis pathway. PINK1 was significantly upregulated in the mutant, however, no significant difference in TH gene expression was observed between groups. We then sought to measure the levels of dopamine in transfected cells using liquid chromatography-mass spectrometry (LC-MS). Although no dopamine was detected using LC-MS, higher levels of phenylalanine, a precursor of dopamine, were observed in the mutant. Using this model, we sought to test the protective effects of curcumin using assays that measure cellular and mitochondrial health. A toxic paraquat concentration of 1.7 mM was chosen to elicit a 50 % decrease in cell viability for the model, while a curcumin concentration of 2.5 μM was chosen as it exhibited no toxic effects. Following the establishment of the model, four treatment groups were established for all experiments thereon: untreated control, curcumin only treatment, paraquat only treatment, and pre-treatment (curcumin treatment followed by paraquat treatment). We found that curcumin was unable to significantly rescue the paraquat-induced reduction in cell viability and mitochondrial membrane potential. The latter was significantly reduced in PINK1 transfected groups, more so in the G309D mutant, indicating the toxic effects of the mutation. Thereafter, the effects of curcumin and polycaprolactone encapsulated nanocurcumin on cell viability were compared. Formulations of curcumin including nanocurcumin are postulated to improve the stability and efficacy of curcumin. Interestingly, curcumin had a greater protective effect, whereas nanocurcumin as well as the empty nanoparticles elicited toxicity. In fact, p re-treatment with the nanocurcumin prior to paraquat treatment caused a 30 % greater loss in cell viability compared to the paraquat treatment alone. Finally, a literature review was published, exploring the potential of consistent dietary consumption of curcumin as an alternative or supplement to existing therapies. We speculate that curcumin binds to α- synuclein protein (found to accumulate in PD) and that this complex is subsequently excreted from the body via the large intestine. In this view, replacing some of the PD drugs in an individual’s treatment regime with a nutraceutical, or ‘functional food’, like curcumin may improve therapeutic benefits with fewer side effects. Considering these results and the published evidence for curcumin as a dietary ‘nutraceutical’, further studies are required to optimize curcumin treatment before advocation of its widespread use as a PD therapeutic agent. Study limitations include the use of an unverified WT plasmid and an undifferentiated cell line, which can be addressed in future work. The findings in this study are of importance as they may contribute to advancing the development of novel plant-based therapies to treat and potentially prevent this detrimental disease.
  • Thumbnail Image
    Item
    Identification of components of turmeric as potential therapeutic agents to slow the progression of neurodegeneration in Parkinson’s disease
    (Stellenbosch : Stellenbosch University, 2022-04) Jansen van Rensburg, Zune; Bardien, Soraya; Kenyon, Colin; Abrahams, Shameemah; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurological disorder associated with severe loss of dopaminergic neurons in the substantia nigra. These neurons are particularly vulnerable due to increased iron and decreased antioxidant levels with aging, constant exposure to reactive oxygen species (ROS), and the presence of neurotoxic compounds. A literature review was compiled and published, in which we postulate that ROS, iron, alpha-synuclein protein (α-syn), and neuromelanin form a toxic feedback loop in individuals with PD. This feedback loop is theorised to be an early trigger culminating in neuronal death and subsequent spread of the disease to neighbouring neurons. Consequently, antioxidants and iron-chelators may be important therapeutic agents to target the accumulated ROS and iron in these neurons. Turmeric is an attractive therapeutic candidate since it has well-established antioxidant properties and may chelate iron. This study aimed to determine which components of turmeric have strong antioxidant and iron-chelating properties that could potentially protect against dopaminergic neuronal degeneration in PD. A crude extract was obtained from purchased turmeric using Soxhlet extraction. Subsequently, the crude extract was separated into different compounds using thin-layer chromatography (TLC). Mass spectrometry (MS) was used to identify the eluted compounds and a selected number of compounds were further analysed with liquid chromatography-mass spectrometry (LCMS). After identifying the compounds, four were chosen to test their total antioxidant capacity and iron chelation ability by comparing these compounds to Trolox (a known antioxidant) and ethylenediaminetetraacetic acid (EDTA) (a strong iron chelator), respectively. Finally, a human neuronal cell line, SH-SY5Y, was used to evaluate the effect of the extracted compounds on cell viability, through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We successfully obtained a crude extract from turmeric powder after Soxhlet extraction. A total of 34 bands were initially eluted from the TLC plate, with the experiment performed in duplicate. After MS, only 15 of the 34 samples had identifiable compounds. Through LCMS, four compounds were identified with confidence namely bis-demethoxycurcumin, demethoxycurcumin, curcumin, and aromatic (ar)-turmerone. The antioxidant assays showed that curcumin had the strongest antioxidant activity while ar-turmerone had the weakest. Analysis of the iron-chelating activity of these compounds found contradictory results. However, with spectrophotometric observation and a commercially validated kit, the curcuminoids were shown to have strong iron-chelation potential. Unexpectedly, ar-turmerone also showed iron-chelation potential albeit to a lesser extent compared to the curcuminoids. The exploratory analysis with the SH-SY5Y cells showed that bis-demethoxycurcumin increased cell viability by 152%, while ar-turmerone decreased it by 73.5%, versus the vehicle control. These promising findings warrant further study using more targeted techniques. Identifying new therapies for PD is of utmost importance since the current treatments only treat the symptoms and do not address the pathobiology of neuronal loss. Natural compounds extracted from plants may be particularly useful for the design of new treatment modalities since they may be more cost- effective and have fewer side effects than synthetic medicines.
  • Thumbnail Image
    Item
    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.