- Title
- The relationship of iron and amyloid: insights from a new mouse model of iron loading and amyloidosis
- Creator
- Aryal, Ritambhara
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Alzheimer’s disease (AD) is a neurodegenerative disease which has been proposed to be associated with brain iron abnormalities, although this remains contentious. To investigate the broad hypothesis that increased brain iron levels may exacerbate Alzheimer’s amyloid pathology, this project studied a novel mouse model (the ‘Aβ+Iron model’). This model was developed by cross-breeding the APPswe/PS1∆E9 mouse model of amyloidosis (the ‘Aβ model’) with the Hfe-/-xTfr2mut mouse model of the iron loading disorder haemochromatosis (the ‘Iron model’) and backcrossing onto the AKR background strain to maximize iron loading. Brain iron content by non-haem iron assay of homogenised brain hemispheres at 6 months of age showed substantial iron loading in Aβ+Iron mice compared to age-, gender- and strain-matched Aβ mice (fold change ≥ 1.8, p<0.0001, n ≥11 mice/group, with ≥4 per sex in each group). Likewise the relative intensity of 3,3'-diaminobenzidine-4HCl (DAB)-enhanced Perls’ staining for iron was significantly increased in Aβ+Iron mice compared to Aβ mice (fold change 1.7, p<0.0001, n=4 mice/group). Since the Iron model does not express human Aβ, it would not be generally predicted to have classical amyloid. This model was used to assess whether abnormal mouse Aβ deposition could be induced by the presence of increased brain iron levels even though this model does not contain any human Aβ sequence. As expected, classical amyloid with Congo red birefringence under polarised light was not observed in the Iron model. These mice produce only endogenous murine Aβ which is not likely to aggregate and form plaques (i.e model does not express human Aβ), suggesting increased brain iron levels alone are not sufficient to induce amyloid formation in the absence of amyloid-related mutations. Histological labelling with Congo red stain for amyloid alone or in combination with DAB-enhanced Perls’ stain for iron was used to examine whether increased brain iron levels altered amyloid deposits in the Aβ and Aβ+Iron transgenic models. No differences were observed for mean counts and size distribution of amyloid deposits, amyloid burden or amyloid density across matched Bregma (-2.46 to -3.16) in Aβ+Iron compared to Aβ mice (all p>0.05, n=4 mice/group). This Bregma range includes hippocampal and entorhinal cortex regions strongly affected in AD. An increased proportion of amyloid plaques had observable iron labelling in the Aβ+Iron model, in which 99.25% of detected plaques co-localised with DAB-enhanced Perls’ stainable iron compared to 81.09% in the Aβ model (p=0.0016, n=4 mice/group). Likewise, iron labelling around plaques was stronger in the Aβ+Iron model than the Aβ model, based on the ImageJ Transformed Mean Gray Value, a measure of staining intensity, of the iron halos surrounding the Congo red plaque cores (fold change 1.4, p=0.0456, n=4 mice/group). However there was no difference in the mean area of the halos surrounding plaques between the two models (p=0.1007, n=4 mice/group). Co-labelling was also performed for iron (DAB-enhanced Perls’ stain) and Aβ peptide using antibody 4G8. This antibody, in addition to classical Aβ amyloid that is birefringent under polarised light, also detects other forms of insoluble Aβ peptide deposits. Antibody 4G8 and iron generally co-localised closely, with detectable iron usually though not always restricted to the 4G8 immunolabelled region. In general, there were more amyloid plaques in brain areas with less iron staining compared to areas with high iron staining (correlation coefficient -0.97). For example, few if any amyloid plaques were detected in basal ganglia and thalamus with strong iron staining compared to regions such as the hippocampus and entorhinal cortex. Overall, regional and cellular distributions of iron in the Aβ+Iron model were similar to those in the Iron model with the important exceptions that iron co-localised with amyloid plaques in the Aβ+Iron model and iron-laden cells were present in the immediate vicinity of plaques. Iron staining was most conspicuous in the choroid plexus by all methods used (DAB-enhanced Perls’ stain and traditional or perfusion Turnbull stain for ferrous iron). Neurons contained very little stainable iron in any region examined, including hippocampus, cerebral cortex and midbrain. Co-labelling with DAB-enhanced Perls’ stain and Luxol fast blue stain or a myelin-specific marker 2’, 3’-cyclic-nucleotide 3’-phosphodiesterase (CNPase) revealed substantial amounts of iron in myelinated regions. Ferritin heavy and light chain immunolabelling co-localised with DAB-enhanced Perls’ stain in a subset of myelin-associated cells with the morphology of oligodendroglial ‘trains’, consistent with previous literature demonstrating iron in a subset of oligodendroglia in rodents and humans. Brain regions with numerous glial fibrillary acidic protein (GFAP) labelled astrocytes typically had few iron-laden cells, with few if any cells co-labelled for both GFAP and iron. Likewise regions with increased iron accumulation typically contained few astrocytes, suggesting there was little reactive astrogliosis in areas with increased iron accumulation, and little if any reactive astrogliosis was observed around amyloid plaques. The proportion of plaques surrounded by iron-laden cells resembling activated amoeboid microglia or microglia transitioning into activation states was much greater in the Aβ+Iron model compared to the Aβ model (22% as opposed to 77%), suggesting iron may exacerbate microgliosis in response to Aβ. This is potentially important since microglial activation appears to facilitate clearance of amyloid, although excessive accumulation of iron could eventually wind up damaging or killing microglia and weakening the brain’s defensive responses. Additional studies are required to investigate these possibilities since few if any cells co-labelled for both iron and the ionized calcium-binding adapter molecule 1 (Iba1), a marker for microglia and while Iba1 positive microglia were present in the immediate vicinity of iron-amyloid complexes, these did not co-label for iron, although reduced sensitivity of double and triple labelling procedures cannot be ruled out. There was no difference in the average count of Iba1-positive microglia in the vicinity of plaques between the Aβ+Iron and Aβ models (p=0.4073, n=4 mice/group). There was limited preliminary evidence of oxidative damage in the Aβ+Iron model. While no labelling was detected in any model for 8-hydroxy-2'-deoxyguanosine (8-OHdG), which detects DNA oxidation damage, there was some putative positive but very weak immunolabelling in all models for lipid peroxidation damage as assessed by 4-hydroxynonenal (4-HNE) antibody that appeared slightly stronger in the Aβ+Iron and Aβ models but this also needs to be confirmed in further studies. Increased levels of iron did not seem to increase neuronal loss in preliminary studies with neuronal nuclear (NeuN) antibody labelling. Specifically there was no significant decrease in relative neuronal counts per unit area at matched Bregma in the Aβ+Iron model compared to the Aβ model in the full cerebral hemisphere, excluding the cerebellum (p=0.3331, n=4 mice/group, one-tailed t test). In summary, these results confirm that brain iron levels are increased in the Aβ+Iron model at 6 months of age and that iron co-localises with amyloid in this model but does not appear to affect measures of amyloid load. Although there was some preliminary evidence of lipid peroxidation damage and increased levels of ferrous iron in a few areas with high levels of iron by DAB-enhanced Perls’ staining, amyloid formation was usually not observed in these regions and no neuronal death was observed across the cerebral hemisphere. Several protective mechanisms may be involved. Most iron appears to remain sequestered within myelin, oligodendroglia or other unidentified glia, with neurons containing little if any Perls’ stainable iron. Cells morphologically resembling transitional or activated amoeboid microglia appear to take up iron in the vicinity of amyloid plaques and may also have protective roles but these were not confirmed to be microglia by Iba1 labelling and remain unidentified. This study has provided new insights into the nature of the relationship of iron and AD. The findings suggest that surplus iron may be safely sequestered by normal brain iron homeostatic and storage mechanisms and may not appreciably influence Alzheimer’s disease pathogenesis at least in the earlier stages of the disease course corresponding to the period examined in the present study. In the light of these observations, the low levels of neuronal iron and the possibility that at least initially, iron may be important in increasing activation of microglia around plaques, facilitating amyloid clearance, iron chelation may be potentially deleterious, at least in the early stages of disease and extreme caution should be exercised before pursuing clinical trials or recommending iron chelation as a treatment for AD and other neurodegenerative conditions.
- Subject
- brain; iron; amyloid; Alzheimer's disease; mouse model
- Identifier
- http://hdl.handle.net/1959.13/1411211
- Identifier
- uon:36313
- Rights
- Copyright 2020 Ritambhara Aryal
- Language
- eng
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