Molecular genetic studies of iron metabolism in Parkinson's disease and related neurodegenerative disorders
Lead Research Organisation:
University of Birmingham
Department Name: Neuroscience
Abstract
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Technical Summary
Dysregulated iron homeostasis occurs in Parkinson‘s disease (PD) and may be central to disease pathogenesis or progression, primarily due to impairment of mitochondrial function. We hypothesize that alteration in the expression of key genes and their resultant proteins involved in neuronal iron homeostasis may underlie neuronal iron dysregulation in PD. We have identified three neuronal proteins for study: the lactoferrin receptor (involved in iron uptake and demonstrated to be increased in PD nigra), mitochondrial ferritin (an iron storage protein that protects mitochondria from iron-mediated oxidative stress) and ferroportin (important for iron export, increased expression protecting neurons from iron-mediated toxicity). The specific aims of this project are:
1. To accurately define the expression of the lactoferrin receptor, ferroportin and mitochondrial ferritin in human post mortem tissue in regions affected by Parkinson‘s disease compared with controls.
· Immunohistochemistry and in situ hybridization studies will be performed using semi-quantitative techniques to determine levels of gene and protein expression in substantia nigra and other regions involved in PD.
· Should differences be found, the study will be extended to include other conditions associated with Lewy body pathology such as Dementia with Lewy bodies.
2. To investigate whether susceptibility to developing PD may be conferred by polymorphisms in genes encoding the three proteins.
· DNA samples will be collected from people with PD and spouse/carer controls (estimated accrual rate of 200 PD and 150 control samples/yr).
· SNPs will be identified, prioritised and genotyped by various methods including allele specific PCR, restriction digestion and sequencing.
· Genotype and allele frequencies will be compared in cases and controls.
3. To investigate the effect of oxidative stress on gene and protein expression in a cell culture system.
· The effects of oxidative stress on expression of the genes and proteins will be assessed in human neuroblastoma cells by real time PCR and Western blotting following exposure of the cells to oxidative stress.
· Levels of gene expression will be modulated using gene knockdown techniques and the effect on cell survival following exposure to oxidative insults assessed.
· Site-directed mutagenesis will be used to assess the functional effects of any disease-associated polymorphisms.
This study will add significantly to our understanding of the role of iron regulation in the pathogenesis of PD. If the mechanisms underlying neuronal degeneration can be identified, it may be possible to intervene therapeutically in patients with early PD to prevent pathological spread and hence disease progression.
1. To accurately define the expression of the lactoferrin receptor, ferroportin and mitochondrial ferritin in human post mortem tissue in regions affected by Parkinson‘s disease compared with controls.
· Immunohistochemistry and in situ hybridization studies will be performed using semi-quantitative techniques to determine levels of gene and protein expression in substantia nigra and other regions involved in PD.
· Should differences be found, the study will be extended to include other conditions associated with Lewy body pathology such as Dementia with Lewy bodies.
2. To investigate whether susceptibility to developing PD may be conferred by polymorphisms in genes encoding the three proteins.
· DNA samples will be collected from people with PD and spouse/carer controls (estimated accrual rate of 200 PD and 150 control samples/yr).
· SNPs will be identified, prioritised and genotyped by various methods including allele specific PCR, restriction digestion and sequencing.
· Genotype and allele frequencies will be compared in cases and controls.
3. To investigate the effect of oxidative stress on gene and protein expression in a cell culture system.
· The effects of oxidative stress on expression of the genes and proteins will be assessed in human neuroblastoma cells by real time PCR and Western blotting following exposure of the cells to oxidative stress.
· Levels of gene expression will be modulated using gene knockdown techniques and the effect on cell survival following exposure to oxidative insults assessed.
· Site-directed mutagenesis will be used to assess the functional effects of any disease-associated polymorphisms.
This study will add significantly to our understanding of the role of iron regulation in the pathogenesis of PD. If the mechanisms underlying neuronal degeneration can be identified, it may be possible to intervene therapeutically in patients with early PD to prevent pathological spread and hence disease progression.