Browsing by Author "Van der Merwe, Celia"

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    Altered mitochondrial respiration and other features of mitochondrial function in parkin-mutant fibroblasts from parkinson’s disease patients
    (Hindawi Publishing Corporation, 2016) Haylett, William; Swart, Chrisna; Van der Westhuizen, Francois; Van Dyk, Hayley; Van der Merwe, Lize; Van der Merwe, Celia; Loos, Ben; Carr, Jonathan; Kinnear, Craig; Bardien, Soraya
    Mutations in the parkin gene are the most common cause of early-onset Parkinson’s disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation.
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    Analysis of copy number variation and disease mechanisms underlying Parkinson’s disease
    (Stellenbosch : Stellenbosch University, 2016-03) Van der Merwe, Celia; Bardien, Soraya; Loos, Ben; 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 movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental and genetic factors. To date, a number of PD-causing genes have been found. The PINK1 gene is of particular interest for this study, and mutations in this gene result in autosomal recessive inheritance of early onset PD. PINK1 plays a vital role in mitochondrial quality control and homeostasis, and in its absence it is thought to result in an accumulation of dysfunctional mitochondria in neurons, culminating in neuronal cell death. Whilst pharmacological and surgical interventions are available for PD, the current options exhibit adverse side effects with long term treatment. There is a great need to develop new treatments with i. less side effects and ii. that can simultaneously target the multiple pathways associated with this disorder. One molecule is curcumin, the core component of the curry spice turmeric, which is well known for its antioxidant and anti-inflammatory properties and has already been studied for its possible neuroprotective role in Alzheimer’s disease. The aim of the present study was to create a cellular model of PD by decreasing the expression of PINK1 in SH-SY5Y neuroblastoma cells. Thereafter, we aimed to test the protective effects of curcumin on this model in the presence and absence of a known stressor, paraquat. This study also aimed to detect possible copy number variation (CNV) in PINK1 (and other PD-causing genes) in a cohort of South African patients with PD, in order to obtain patient-derived fibroblasts to verify the results obtained from the original cellular model. PINK1 was knocked down using siRNA (Qiagen, USA) in SH-SY5Y neuroblastoma cells, and the knock down was verified by quantitative real time PCR (qRTPCR) and western blotting. Thereafter, PINK1 siRNA cells and control cells were separated into four treatment groups: i. untreated, ii. treated with 25μM paraquat for 24hours, iii. pre-treated with 2μM curcumin for 1hour then treated with 25μM paraquat for 24hours, or iv. treated with 2μM curcumin for 1hour, and various parameters of cellular and mitochondrial function were measured. Cell viability was measured by an MTT assay. Western blot analysis was performed using cleaved PARP and full-length caspase 3 markers to detect levels of apoptosis, and LC3-II and p62 markers to detect autophagic flux. Mitochondrial respiration experiments were completed on the Seahorse XF Analyser using the Mito Stress Test Kit and the Glycolysis Stress Test. Flow cytometry was utilised to measure mitochondrial membrane potential (MMP) using the JC- 1 fluorochrome, and mitochondrial network was analysed by fluorescent microscopy. For CNV detection, MLPA was performed on 210 South African PD patients and putative mutations were verified by qRTPCR on the Lightcycler 96. PINK1 was successfully knocked down at a gene and protein expression level. The PINK1 siRNA cells exhibited a significant decrease in cell viability (p=0.0036), and an increase in apoptosis (p=0.0144). A decrease in PINK1 expression also resulted in significantly decreased MMP (p=0.0008), mitochondrial respiration (p=0.0015), ATP production (p=0.002) and glycolytic capacity (p=0.0445). No significant changes were observed in the connectivity of the mitochondrial network, but autophagic flux was significantly increased in the PINK1 siRNA cells, as detected by increased LC3-II levels (p=0.0152). As expected, paraquat-treated cells exhibited decreased cell viability, increased apoptosis, decreased MMP, autophagic flux, and a more fragmented mitochondrial network. Paraquat treatment therefore successfully acted as a stressor on the cells. Curcumin pre-treatment followed by paraquat treatment rescued cell viability in control cells (p=0.003), and significantly decreased apoptosis in PINK1 siRNA cells (p=0.0018). Curcumin protected mitochondrial dysfunction in PINK1 siRNA cells by increasing MMP (p=0.0472) and maximal respiration (p=0.0014), as well as significantly increasing MMP (p=0.0307) and maximal respiration (p=0.032) in control cells. Additionally, curcumin treatment resulted in increased autophagic flux (p=0.0017) in stressed control cells. These results highlight a protective effect of curcumin against paraquat and against the damaging effects on the mitochondria in cells with decreased PINK1 expression. Lastly, MLPA analysis did not reveal any PINK1 CNV mutations in a total of 210 South African PD patients, and fibroblasts were therefore not obtained. A number of false positive mutations were identified that were not verified by qRTPCR. A common polymorphism M192L resulting in a false positive PARK2 exon 5 deletion was found in a number of patients, all of whom were of Black or Mixed Ancestry ethnic groups. One patient was shown to harbour a heterozygous deletion in PARK2 exon 4. In conclusion, PINK1 siRNA-mediated knock down in SH-SY5Y neuroblastoma cells can be used as a model of PD to study aspects of mitochondrial dysfunction. Furthermore, curcumin should be considered as a possible therapeutic target for PD, as it exhibits protective effects against paraquat at a mitochondrial level. Given the low toxicity of curcumin, and the fact that it is already part of a dietary regimen in most populations worldwide, further studies on elucidating its biochemical and cellular properties are therefore warranted. The use of natural compounds such as curcumin as therapeutic agents is currently a topical and fast-growing area of research, and holds much promise for clinical application in various diseases including neurodegenerative disorders such as Alzheimer’s disease and PD.
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    Factors influencing the development of early- or late-onset Parkinson's disease in a cohort of South African patients
    (Health & Medical Publishing Group, 2012-10-01) Van der Merwe, Celia; Haylett, William; Harvey, Justin; Lombard, Debbie; Bardien, Soraya; Carr, Jonathan
    Background. Neurodegenerative disorders such as Parkinson’s disease (PD) contribute significantly to global disease burden. PD can be categorised into early-onset PD (EOPD) with an age at onset (AAO) of ≤50 years and late-onset PD (LOPD) with an AAO of 50 years. Aims. To identify factors influencing EOPD and LOPD development in a group of patients in South Africa (SA). Methods. A total of 397 unrelated PD patients were recruited from the Movement Disorders Clinic at Tygerberg Hospital and via the Parkinson’s Association of SA. Patient demographic and environmental data were recorded and associations with PD onset (EOPD v. LOPD) were analysed with a Pearson’s Chi-squared test. The English- and Afrikaans-speaking (Afrikaner) white patients were analysed separately. Results. Logistic regression analysis showed that ethnicity (p<0.001) and family history (p=0.004) were independently associated with AAO of PD. Average AAO was younger in black, coloured and Afrikaner patients than English-speaking white patients. A positive family history of PD, seen in 31.1% of LOPD patients, was associated with a younger AAO in the study population. Conclusions. These associations may be attributed to specific genetic and/or environmental risk factors that increase PD susceptibility and influence the clinical course of the disorder. More studies on PD in the unique SA populations are required to provide novel insights into mechanisms underlying this debilitating condition.
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    An investigation into the role of mitochondrial dysfunction in South African Parkinson’s disease patients
    (Stellenbosch : Stellenbosch University, 2012-12) Van der Merwe, Celia; Bardien, Soraya; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
    ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental (such as exposure to pesticides and neurotoxins) and genetic factors. A number of PD-causing genes have been found including SNCA, LRRK2, EIF4G1 and VPS35 (for autosomal dominant forms of PD) and parkin, PINK1, DJ-1 and ATP13A2 (for autosomal recessive forms of PD – arPD). Mutations in the parkin gene are the predominant cause of arPD. Parkin plays a role in the ubiquitin-proteasomal system which degrades damaged and unwanted proteins in the cell and it is also thought to be involved in maintaining healthy mitochondria. Numerous studies have implicated mitochondrial function in the pathogenesis of PD. Therefore the aim of the present study was to investigate the role of mitochondrial dysfunction in PD patients with parkin-null mutations. Four South African PD patients, each harbouring two parkin-null mutations, were recruited for this study. A muscle biopsy was performed for analysis of mitochondrial morphology using histology and transmission electron microscopy (TEM). Skin biopsies were taken, from which fibroblasts were cultured. These fibroblasts were used in i) mitochondrial morphological assessments using TEM, ii) mitochondrial network analysis, iii) functional studies via ROS measurement and iv) analysis of the proteome using a LTQ Orbitrap Velos mass spectrometer. In addition, RNA was isolated from peripheral blood samples for gene expression studies using the RT² Profiler PCR Array (SABiosciences, USA) and the RT² PCR Primer Assay (SABiosciences, USA). Heterozygous family members (carriers) and wild-type controls were also recruited for this study. Results from the histological and TEM analysis from the muscle biopsy observed subtle mitochondrial changes including the presence of type II fibres, atrophic fibres, the presence of lipids, and wrinkling of the sarcolemmal membrane. Enlarged mitochondria were also observed in one patient. TEM analysis on the patient’s fibroblasts observed an increase in the number of electron dense vacuoles, speculated to be autolysosomes. The mitochondrial network in two of the patients’ fibroblasts showed fragmented and dot-like networks which are indicative of damaged mitochondria. An increase in mitochondrial ROS levels was observed in three of the four patients. Expression studies found down-regulation of 14 genes from four of the five mitochondrial complexes and a total of 688 proteins were found only in the control and not in the patient fibroblasts. Some of these proteins are known to be part of the ‘mitochondrial dysfunction’ pathway. Taken together, these results indicate that the absence of parkin results in a number of mitochondrial alterations. Based on these findings, a model of PD was proposed: It is speculated that when parkin is absent, electron transport chain complex genes are down-regulated. This results in impaired oxidative phosphorylation, causing an increase in the production of mitochondrial ROS and subsequent oxidative stress. Mitochondria are then damaged; resulting in the fragmentation of the mitochondrial network. The impaired mitochondria are thus tagged for degradation, causing the recruitment of autolysosomes which engulf the mitochondria via mitophagy. Ultimately, as the compensatory mechanisms fail, this triggers the consequential cascade of cellular apoptotic events. This study has elucidated the effect of parkin on the mitochondria, and can act as a ‘stepping stone’ towards future development of therapeutic strategies and/or biochemical markers that will benefit not only patients with PD but also other neurodegenerative disorders.