- Title
- Cellular and molecular changes in the brain of the Hfe-/-xTfr2mut mouse, a model of human iron loading
- Creator
- Heidari, Moones
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2016
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Brain iron dyshomeostasis has been proposed to be associated with various severe neurodegenerative diseases such as Alzheimer’s disease, although the nature of the association remains controversial. It is unclear whether brain iron accumulation and consequent degeneration are also a feature of peripheral iron loading disorders such as hereditary hemochromatosis. To help understand how systemic iron loading affects the brain, this project studied mice with disruption of two iron regulatory genes, the hemochromatosis (Hfe) gene and the transferrin receptor 2 (Tfr2) gene. Brain iron content measured by non-heme iron assay at 3 or 9 months of age showed significant iron accumulation in the Hfe-/-xTfr2mut mice compared with age-, gender- and strain-matched wildtype mice (fold change ≥1.9, p<0.0005 n≥4 mice/group). Regional iron distribution was investigated by enhanced 3,3'-diaminobenzidine-4HCl (DAB) Perls’ staining, which confirmed greater iron accumulation in the brain of Hfe-/-xTfr2mut mice at 3, 6, 9 and 12 months of age than in the brain of wildtype mice, although the regional and cellular distributions were very similar in both groups of mice. The choroid plexus was the most intensely iron-stained structure. Co-staining with DAB-enhanced Perls’ stain for iron and Luxol Fast Blue stain for myelin showed that in both Hfe-/-xTfr2mut and wildtype mice, substantial proportions of iron were co-localized in myelinated fibers and patches in the corpus callosum, internal capsule, fornix system, basal ganglia and cerebellar white matter. A few cerebellar Purkinje cells occasionally showed very low levels of iron staining that did not appear to differ between Hfe-/-xTfr2mut and wildtype mice. No other neurons, including hippocampal neurons, were observed to show iron loading by DAB-enhanced Perls’ stain in any mice. Co-labeling with specific cell marker antibodies and DABenhanced Perls’ staining revealed that iron was mostly accumulated in a subset of oligodendrocytes and some unidentified cells while neurons, astrocytes and microglia did not show clearly visible co-labeling with iron. Gene expression microarray analysis revealed numerous transcriptome changes, including significant increases (fold change >1.28, p<0.02, n≥5) in brain transcripts for important immediate-early transcription regulators such as FBJ osteosarcoma oncogene (Fos), Jun B proto-oncogene (Junb), early growth response genes 1, 2 and 4 (Egr1, 2 and 4) and decreased transcripts for the transcription repressor Zfp68. These expression changes in key transcription factors are consistent with the numerous transcriptome changes observed and are likely to influence the downstream expression of multiple genes, affecting numerous brain systems. Some of these changes appear likely to be compensatory responses while others may reflect perturbations of brain systems. Several important genes related to iron metabolism showed decreased brain transcript levels, including the genes for transferrin (Tf), transferrin receptor 1 (Tfr1), ceruloplasmin (Cp) and hepcidin (Hamp). The decrease in Tfr1 transcripts and high ferritin protein levels, described elsewhere (Heidari et al. 2015), are cellular responses typically seen with increased intracellular iron loading in other systems. These changes support increased iron storage in ferritin and reduced iron up take due to lower transferrin receptor expression. The reduction in transcripts for Tf and Cp, which encode two circulating extracellular proteins, may tend to decrease the amount of iron delivered from cerebral vascular endothelial cells into the brain and ultimately to neurons. All these changes are predicted to maintain brain iron homeostasis and help protect the brain against damage from high iron concentrations. There were increased brain transcript levels for the hemoglobin alpha, adult chain 1 (Hbaa1) and both the hemoglobin beta, adult major and minor chains (Hbb-b1, Hbb-b2), possibly corresponding to neuronal hemoglobin expression, which may also have neuroprotective roles but could also cause tissue damage if released into the extracellular space. Pathway analysis found significant alterations for a group of genes involved in the disease class Neurodegeneration with Brain Iron Accumulation (NBIA). These changes included increased level of ferritin protein (Heidari et al. 2015), reduced levels of transcripts for phospholipase A 2 group VI, fatty acid 2-hydroxylase, ceruloplasmin, chromosome 19 open reading frame 12 and ATPase type 13A2 (fold change >1.17, p<0.04, n≥5 mice/group). Apart from the ferroxidase ceruloplasmin, all the other NBIA genes showing expression changes in response to iron were found to be either involved in myelin homeostasis or to contain mutations that result in demyelination in human patients or both. Further microarray data mining revealed that 18 other myelin-related genes also were down-regulated (p<0.05). This suggests that increased iron levels in the brain might exert effects on NBIA pathology through myelin related systems. Brain transcriptome changes in the Hfe-/-xTfr2mut mouse were then compared with expression profiles of normal human basal ganglia and brains from NBIA patients. Chi square testing showed significant overlap (p<0.0001) of differentially expressed genes in the Hfe-/-xTfr2mut brain with human brain gene networks that exhibited co-expression of NBIA and myelin-related genes. This suggests that increased iron loading selectively influences expression of a set of potentially interrelated NBIA and myelin genes. There was also overlap (p<0.0001) of genes differentially expressed in the Hfe-/-xTfr2mut mouse brain and post-mortem NBIA basal ganglia. Pathway analysis showed that significant enrichment of ‘myelin sheath’ and various other myelin-related ontologies (all p<0.05). This suggests analogies between molecular mechanisms affected by brain iron loading in mice and pathogenic mechanisms in NBIA patients and provides further evidence for involvement of systems relating to myelin. Collectively, these findings have confirmed the presence of higher brain iron content in the Hfe-/-xTfr2mut mice, as hypothesized, and provided evidence that this is primarily localized in myelinated structures, some oligodendrocytes and other still unidentified cells. The project also demonstrated using two experimental techniques (microarray, real time RTPCR) that, as hypothesized, this increased brain iron loading was accompanied by altered expression of iron-related genes, that may protect the brain by increasing iron storage and decreasing iron uptake. Finally altered expression of NBIA genes related to myelin and other myelin-related genes, and correspondence between mouse transcriptome changes, human NBIA and myelin gene networks supports the hypothesis that the changes in iron lead to changes in gene expression relating to molecular systems involved in neurodegenerative disease. The results further provide evidence for the new hypotheses that myelin-associated iron accumulation may occur in hemochromatosis and that this may drives alterations in molecular systems involved in NBIA neuropathogenesis. The research may shed light on potential interrelationships of iron and myelin in the brain in NBIA and other iron disorders, as well as in patients with psychiatric disorders or with myelin disorders such as multiple sclerosis.
- Subject
- brain; myelin; iron; heamochromatosis; neurodegeneration with brain iron accumulation; microarray; histology
- Identifier
- http://hdl.handle.net/1959.13/1312991
- Identifier
- uon:22505
- Rights
- Copyright 2016 Moones Heidari
- Language
- eng
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