Evaluating the neuroprotective effect of curcumin on a PINK1 cell model of Parkinson's disease

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dc.contributor.advisorBardien, Sorayaen_ZA
dc.contributor.advisorKenyon, Colinen_ZA
dc.contributor.advisorAbrahams, Shameemahen_ZA
dc.contributor.authorChetty, Devinaen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.en_ZA
dc.date.accessioned2022-02-20T07:40:44Z
dc.date.accessioned2022-04-29T09:16:45Z
dc.date.available2022-02-20T07:40:44Z
dc.date.available2022-04-29T09:16:45Z
dc.date.issued2022-04
dc.descriptionThesis (MSc)--University of Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH 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.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is 'n neurodegeneratiewe versteuring wat gekenmerk word deur 'n verlies aan neurone wat die neurotransmitter dopamien produseer. Merkwaardig, ten spyte van omrykende studies wat talle gedisreguleerde prosesse verwant aan PS geopenbaar het, bestaan fundamentele gapings steeds in ons kennis van die siektepatofisiologie. Ons begrip van die siekte sluit prosesse soos mitochondriale disfunksie in wat verhoogde oksidatiewe stres en energiemislukking veroorsaak, asook verkeerde proteïenophoping in groot insluitings. Die tydruimtelike volgorde van gebeure wat tot PS lei en, belangriker, die aanvanklike faktore wat siekte-aanvang veroorsaak, bly egter ontwykend. Dit daag die ontwikkeling van effektiewe terapeutiese strategieë uit om PS te voorkom en te genees. Bowendien, het die huidige konvensionele farmaseutiese behandelinge vir PS tale newe- effekte. Dit dien as motivering vir soektogte na ‘n natuurlike behandeling wat die aanvullende leiding kan verminder van ‘n reeds afbrekende siekte. Dit is duidelik dat mitochondriale disfunksie en oksidatiewe stres betrokke is by die siekte se patologie, daarom kan die bestudering van hierdie verskynsels in PS lei tot die ontwikkeling van meer effektiewe terapeutiese strategieë. Kurkumien is 'n plantgebaseerde polifenol wat waargeneem is om antioksidant eienskappe te toon, verhoog sel lewenskragtigheid en verbeter mitochondriale funksie. Gevolglik was die doel van die huidige studie om 'n PS-sellulêre model te skep en die potensiële beskermende effekte van kurkumien te evalueer. Daarom het ons probeer om 'n toepaslike PS-model te vestig deur SH-SY5Y-selle met wilde-tipe (WT) of G309D muteerde PINK1 cDNA te transreguleer, waarvan laasgenoemde dopamien op gereguleer het wat gelei het tot sitotoksiteit. Daarbenewens, aangesien verskeie neurotoksiene getoon is om PS te aktiveer, is die giftige onkruiddoder, parakwat, toegedien om die model aan te vul met sellulêre skade en mitochondriale disfunksie. Om die model te bevestig, het ons RT-qPCR uitgevoer om geen-uitdrukkingsvlakke van PINK1 en tyrosine hydroxylase (TH), 'n ensiem betrokke by die chemiese samestelling van dopamien, kwantitatief te meet. PINK1 was aansienlik opgereguleer in die muteerde, maar geen beduidende verskil was opgemerk in TH-geenuitdrukking tussen groepe nie. Ons het toe probeer om die vlakke van dopamien in transfekte selle te meet met behulp van vloeibare chromatografie-massaspektrometrie (VC-MS). Alhoewel geen dopamien opgespoor was met behulp van VC-MS nie, was hoër vlakke van fenylalanien, 'n voorloper van dopamien, in die muteerde waargeneem. Met behulp van hierdie model het ons probeer om die beskermende effekte van kurkumien te toets met behulp van eksperimente wat sellulêre en mitochondriale gesondheid kan bepaal. 'n Giftige parakwat konsentrasie van 1.7 mM was gekies om 'n 50% afname in sel lewensvatbaarheid in die model te ontlok, terwyl 'n kurkumien konsentrasie van 2.5 μM gekies was, aangesien dit geen giftige effekte getoon het nie. Na die bevestiging van die model was vier behandelingsgroepe gestig vir alle eksperimente wat volg daarna, dit sluit in: onbehandelde kontrole, kurkumien alleenlik, parakwat alleenlik, en voor-behandeling (kurkumien behandeling gevolg deur parakwat behandeling). Ons het gevind dat kurkumien nie merkwaardig die selle kon beskerm teen die verlaagde sel lewenskragtigheid en mitochondirale membraanpotentiaal wat geïnduseer was deur parakwat nie. Die mitochondriale membraanpotentiaal was aansienlik verminder in die PINK1-getransfekteerde groepe, meer so as in die G309D-muteerde, dit dui die giftige effekte van die mutasie aan. Daarna is die effek van kurkumien en policaprolactone ingeslote nanokurkumien op sel lewensvatbaarheid vergelyk. Formulerings van kurkumien, insluitend nanokurcumin, word gepostuleer om die stabiliteit en doeltreffendheid van kurkumien te verbeter. Interessant genoeg, kurkumien het 'n groter beskermende effek gehad, terwyl nanokurcumin sowel as die leë nanopartikels toksisiteit getoon het. In werklikheid, het die voorbehandeling met nanokurkumien (voor parakwat behandeling) ‘n 30% groter verlies in sel lewenskragtigheid veroorsaak as die selle met slegs parakwat behandeling. Ten slotte is 'n literatuuroorsig gepubliseer, wat die potensiaal van konsekwente dieet verbruik van kurkumien ondersoek as 'n alternatief of aanvulling tot bestaande terapieë. Ons spekuleer dat kurkumien aan α- sinukleien proteien bind (gevind om te versamel in PS) en dat hierdie kompleks daarna uit die liggaam uitgeskei word deur die dikderm. Deur dit in ag te neem, kan die vervanging van sommige van die huidige PS-behandelinge met nutraceutiese produkte, of ‘funksionele kos’, soos kurkumien, voordelig wees en minder newe-effekte hê. Samevattend met hierdie resultate en die gepubliseerde bewyse vir kurkumien as ‘n dieet ‘nutraceutical’, is verdere studies nodig om kurkumien behandeling te optimiseer voordat die wydverspreide gebruik daarvan as 'n PS terapeutiese middel kan gebeur. Studie beperkings sluit in die gebruik van 'n ongeverifieerde WT-plasmied en 'n ongedifferensieerde sellyn, wat in toekomstige werk aangespreek kan word. Die bevindings in hierdie studie is van belang, aangesien dit kan bydra tot die bevordering van die ontwikkeling van nuwe plantgebaseerde terapieë om hierdie nadelige siekte te behandel en moontlik te voorkom.af_ZA
dc.format.extent155 pagesen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/124503
dc.language.isoen_ZAen_ZA
dc.publisherStellebosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectParkinson's diseaseen_ZA
dc.subjectCurcuminen_ZA
dc.subjectNeutraceuticalen_ZA
dc.subjectNeurodegenerative disorderen_ZA
dc.subjectUCTDen_ZA
dc.titleEvaluating the neuroprotective effect of curcumin on a PINK1 cell model of Parkinson's diseaseen_ZA
dc.typeThesisen_ZA
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