The contribution of an iron genetic modifier, HFE, to Alzheimer's disease
Open Access
- Author:
- Hall II, Eric Christopher
- Graduate Program:
- Neuroscience
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 17, 2009
- Committee Members:
- James Robert Connor, Dissertation Advisor/Co-Advisor
James Robert Connor, Committee Chair/Co-Chair
Anne M Andrews, Committee Member
Robert J Milner, Committee Member
Jiyue Zhu, Committee Member
Jack T Rogers, Committee Member - Keywords:
- amyloid
neurodegeneration
HFE
iron
tau
pin1 - Abstract:
- Alzheimer’s disease (AD) is a neurodegenerative disorder of the human central nervous system characterized by loss of memory that leads to dementia. The pathological characteristics of AD include an accumulation of amyloid-â (Aâ) plaques and aggregated hyperphosphorylated tau protein, neurofibrillary tangles (NFT), throughout the brain. Multiple hypotheses have been proposed to explain AD etiology including the loss of brain iron regulation. Among the areas investigated are the of variant forms of the HFE gene as a risk factor or disease modifier. HFE is an iron regulatory protein that limits cellular iron uptake. A mutation in the HFE gene can result in a loss of function. Epidemiological studies have investigated an association between HFE polymorphisms and AD risk; however these studies are not all in agreement. In order to determine more clearly how HFE could impact neurodegenerative processes, experiments in this thesis were designed to investigate the cellular contribution of the HFE H63D polymorphism in proposed AD pathogenic pathways. We utilized a novel stably transfected human neuroblastoma SH-SY5Y cell model expressing HFE polymorphisms for our studies. The thesis focused on three main areas: 1) HFE and amyloid regulation, 2) HFE and tau phosphorylation and 3) HFE effects on potential drug treatment strategies in AD. To evaluate amyloid regulation, we determined amyloid precursor protein (APP) synthesis, processing, and cellular vulnerability to Aâ toxicity in the presence of different forms of HFE. APP levels increased with HFE expression, although no effect of HFE variants was observed. There was no change in APP processing as determined by performing spectrofluorometric secretase-specific activity assays. We discovered that cells expressing the H63D variant are more sensitive to Aâ peptide exposure determined by MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]. There was an up-regulation of the intrinsic apoptotic pathway as determined by increases in caspase-9 expression, caspase-3 activity, and early apoptosis based on detection of Annexin V. It appeared that these changes were a result of mitochondria dysfunction in the cells expressing H63D because we also observed an increase in Bax in the cellular mitochondrial fraction and cytochrome C levels were increased in the cytosolic fraction. Mitochondria dysfunction and oxidative stress can act as indirect mechanisms to impact tau phosphorylation. Tau phosphorylation is regulated by a homeostasis of tau kinase and phosphatase activity that can be impacted by cellular stressors. In cells expressing the H63D polymorphism, tau phosphorylation was increased at serine-residues implicated in NFT generation. There was no change in phosphatase expression in H63D cells; yet there was an increase in glycogen synthase kinase-3 beta (GSK-3â) activity as determined by measuring GSK-3â phosphorylation at its serine-9 residue. The genotype associated alterations in GSK-3â activity with HFE expression prompted us to investigate the role of a novel intracellular regulator of amyloid and tau phosphorylation; the prolyl-peptidyl cis/trans isomerase, Pin1. Oxidative stress has been shown to impact Pin1 expression and activity, thus we hypothesized that H63D cells would have altered Pin1 activity. Total Pin1 expression levels were not affected by HFE expression; however there was an increase in phosphorylation of Pin1 at its serine-16 residue suggesting a decrease in Pin1 activity in H63D cells. We also found that iron-mediated oxidative stress could increase Pin1 phosphorylation. Overall, these data suggest that gene-environment interactions are significant in elucidating disease etiology and identifying therapeutic targets to improve disease outcomes. Independent of HFE status, we discovered that GSK-3â could be impacted by cellular iron. Thus, increased dietary iron intake could result in even higher GSK-3â activity in individuals carrying an H63D allele. GSK-3â is involved in numerous cellular processes including, but not limited to: protein phosphorylation, mitochondria function, and apoptosis, which were impacted by expression of the H63D HFE polymorphism. These data suggest GSK-3â as a potential pharmacological target that could greatly improve cellular function in AD patients, especially those carrying the H63D allele.