Analysis of copy number variation and disease mechanisms underlying Parkinson’s disease

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 JC1 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). Stellenbosch University https://scholar.sun.ac.za