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The role of melatonin on brain iron homeostasis and its relation to Parkinson’s Disease
thesisposted on 23.02.2017, 23:40 by Lai, Hien Tet
Parkinson’s Disease (PD) is the second most common neurological disorder after Alzheimer’s Disease. The disease will manifest with the loss of up to 70% of the dopaminergic neurons in the Substantia Nigra (SN) region and it mainly affects the elderly. One of the common pathological hallmarks for PD is the excessive iron accumulation within the SN region of the brain. However, the pathogenesis for of the excessive iron levels accumulation in the SN is unclear. The increase in intracellular oxidative stress may play a role in disrupting neuronal iron homeostasis by increasing the expression of iron intake proteins and decreasing expression of iron exporter. Furthermore, iron itself increases reactive oxygen species formation via the Fenton Reaction. It was found that there is a possible link between PD and melatonin, a neurohormone secreted by the pineal gland of the brain. Firstly, there is a decreased in melatonin receptor expression in the brain of PD patients. Furthermore, serum melatonin levels decreased as people age, which could be a link on why PD tend to affect the elderly. Hence the purpose of these study is to elucidate the link between PD and melatonin. The neurohormone itself is a well-studied antioxidant. The decrease in melatonin secretion and receptor could play a role increasing the dopaminergic neuron susceptibility to oxidative stress. The increase of oxidative stress could trigger for disrupting neuronal iron homeostasis. In this thesis, the human neuroblastoma cell line SH-SY5Y was used as an in vitro model for dopaminergic neurons to study the effects melatonin on iron overloaded SH-SY5Y cells. Using qRT-PCR to determine the gene expression of iron transport proteins DMT1, TfR, LfR and FPN1. The protein expression were determined using Cell based ELISA (CBE). It was found that iron loading increases the gene expression of all four iron transport proteins. The protein expression level of the same iron transport proteins was increase similarly. The increased in iron uptake protein expression resulted in increased neuronal iron content after ICP-MS analysis. Melatonin as an anti-oxidant and anti-inflammation attenuated the increase in both the gene and protein expression of the iron transport proteins induced by iron loading. This resulted in decreased intra-neuronal iron levels groups given both iron and melatonin treatment. By using MTT assay to determine cell viability, iron loading induces cell death up to 25% and through cell cycle analysis, it supported the observation that iron loading induced cellular apoptosis. Although, there is minimal improvement in cell viability in the groups given both iron and melatonin. But the findings of cell cycle analysis, showed that melatonin successfully reduced cellular apoptosis in comparison to iron treated group. The increased neuronal apoptosis from iron loading is mediated by the activation components of the MAPK pathway, including both C-Raf and p-C-Raf and p38 kinase. Moreover, the protein expression of NF-κB increased from iron loading, which indicates that iron loading is pro-inflammatory. The increase oxidative stress and inflammation signalling played a role in causing iron homeostasis dysregulation. Co-treatment with melatonin were successful in ameliorating the expression of MAPK markers, C-Raf, p-C-Raf and p38 kinase, along with NF-κB.