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Open Access
Abstract: Iron (Fe) is an essential trace element required for healthy brain function. Yet, disrupted iron neurochemistry, and the associated formation of aberrantly aggregated protein lesions has been implicated in the development of multiple degenerative brain disorders including Alzheimer's disease (AD). Here, nanoscale resolution soft X-ray spectromicroscopy is used to examine the interaction of β-amyloid (Aβ), a peptide fundamentally implicated in the development of Alzheimer's, and ferric (Fe3+) iron. Crucially, by probing the carbon K (280–320 eV) and iron L2,3 (700–740 eV) edges, both the organic and inorganic (iron) sample chemistry was established. The co-aggregation of Aβ and iron is known to influence iron chemistry, resulting in the chemical reduction of Fe3+ into reactive and potentially toxic ferrous (Fe2+) and zero-oxidation (Fe0) states. Here, nanoscale (i.e. sub-micron) variations in both iron oxidation state and the organic composition of Aβ were observed, replicating in vitro the diverse iron chemistry documented in amyloid plaques from human brain, with the chemical state of iron linked to the conformation state of Aβ. Furthermore, aggregates were formed that were morphologically and chemically distinct dependent on the treatment of Aβ prior to the addition of ferric iron. These findings support the hypothesis that Aβ is responsible for altering iron neurochemistry, and that this altered chemistry is a factor in neurodegenerative processes documented in AD. The methods applied here, combining nanoscale-resolution imaging and high chemical sensitivity, enabled discovery of the nanoscale heterogeneity in the iron and carbon chemistry of in vitro aggregates, and these approaches have scope for wider application in metallomics.
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Mar 2021
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I08-Scanning X-ray Microscopy beamline (SXM)
I10-Beamline for Advanced Dichroism
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Open Access
Abstract: Atypical low-oxidation-state iron phases in Alzheimer’s disease (AD) pathology are implicated in disease pathogenesis, as they may promote elevated redox activity and convey toxicity. However, the origin of low-oxidation-state iron and the pathways responsible for its formation and evolution remain unresolved. Here we investigate the interaction of the AD peptide β-amyloid (Aβ) with the iron storage protein ferritin, to establish whether interactions between these two species are a potential source of low-oxidation-state iron in AD. Using X-ray spectromicroscopy and electron microscopy we found that the co-aggregation of Aβ and ferritin resulted in the conversion of ferritin’s inert ferric core into more reactive low-oxidation-states. Such findings strongly implicate Aβ in the altered iron handling and increased oxidative stress observed in AD pathogenesis. These amyloid-associated iron phases have biomarker potential to assist with disease diagnosis and staging, and may act as targets for therapies designed to lower oxidative stress in AD tissue.
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Jun 2020
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[15230]
Open Access
Abstract: Background: Neuromelanin-pigmented neurons, which are highly susceptible to neurodegeneration in the Parkinson’s disease substantia nigra, harbour elevated iron levels in the diseased state. Whilst it is widely believed that neuronal iron is stored in an inert, ferric form, perturbations to normal metal homeostasis could potentially generate more reactive forms of iron capable of stimulating toxicity and cell death. However, non-disruptive analysis of brain metals is inherently challenging, since use of stains or chemical fixatives, for example, can significantly influence metal ion distributions and/or concentrations in tissues. Aims: The aim of this study was to apply synchrotron soft x-ray spectromicroscopy to the characterisation of iron deposits and their local environment within neuromelanin-containing neurons of Parkinson’s disease substantia nigra. Methods: Soft x-ray spectromicroscopy was applied in the form of Scanning Transmission X-ray Microscopy (STXM) to analyse resin-embedded tissue, without requirement for chemically disruptive processing or staining. Measurements were performed at the oxygen and iron K-edges in order to characterise both organic and inorganic components of anatomical tissue using a single label-free method. Results: STXM revealed evidence for mixed oxidation states of neuronal iron deposits associated with neuromelanin clusters in Parkinson’s disease substantia nigra. The excellent sensitivity, specificity and spatial resolution of these STXM measurements showed that the iron oxidation state varies across sub-micron length scales. Conclusions: The label-free STXM approach is highly suited to characterising the distributions of both inorganic and organic components of anatomical tissue, and provides a proof-of-concept for investigating trace metal speciation within Parkinson’s disease neuromelanin-containing neurons.
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May 2020
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I08-Scanning X-ray Microscopy beamline (SXM)
I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[15230, 15854, 20809, 24526, 24531]
Open Access
Abstract: A hallmark of Parkinson’s disease is the death of neuromelanin‐pigmented neurons, but the role of neuromelanin is unclear. Lack of a neuromelanin‐specific marker was highlighted over 30 years ago, yet in‐situ characterization of neuromelanin remains dependent on detectable pigmentation, rather than direct quantification of neuromelanin. We show that direct, label‐free nanoscale visualization of neuromelanin and associated metal ions in human brain tissue can be achieved using synchrotron Scanning Transmission X‐ray Microscopy (STXM), via a characteristic feature in the neuromelanin x‐ray absorption spectrum at 287.4 eV that is also present in iron‐free and iron‐laden synthetic neuromelanin. This is confirmed in consecutive brain sections by correlating STXM neuromelanin imaging with silver nitrate‐stained neuromelanin. Analysis suggests that the 1s ‐ σ* (C‐S) transition in benzothiazine groups accounts for this feature. This advance in visualizing neuromelanin illustrates the wider potential of STXM as a label‐free spectromicroscopy technique applicable to both organic and inorganic materials.
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Mar 2020
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[15854, 19779]
Open Access
Abstract: Transition metals have essential roles in brain structure and function, and are associated with pathological processes in neurodegenerative disorders classed as proteinopathies. Synchrotron x-ray techniques, coupled with ultrahigh-resolution mass spectrometry, have been applied to study iron and copper interactions with amyloid β (1–42) or α-synuclein. Ex vivo tissue and in vitro systems were investigated, showing the capability to identify metal oxidation states, probe local chemical environments, and localize metal-peptide binding sites. Synchrotron experiments showed that the chemical reduction of ferric (Fe3+) iron and cupric (Cu2+) copper can occur in vitro after incubating each metal in the presence of Aβ for one week, and to a lesser extent for ferric iron incubated with α-syn. Nanoscale chemical speciation mapping of Aβ-Fe complexes revealed a spatial heterogeneity in chemical reduction of iron within individual aggregates. Mass spectrometry allowed the determination of the highest-affinity binding region in all four metal-biomolecule complexes. Iron and copper were coordinated by the same N-terminal region of Aβ, likely through histidine residues. Fe3+ bound to a C-terminal region of α-syn, rich in aspartic and glutamic acid residues, and Cu2+ to the N-terminal region of α-syn. Elucidating the biochemistry of these metal-biomolecule complexes and identifying drivers of chemical reduction processes for which there is evidence ex-vivo, are critical to the advanced understanding of disease aetiology.
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Oct 2019
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I08-Scanning X-ray Microscopy beamline (SXM)
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Aug 2018
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[15854, 19779]
Open Access
Abstract: Altered metabolism of biometals in the brain is a key feature of Alzheimer’s disease, and biometal interactions
with amyloid-β are linked to amyloid plaque formation. Iron-rich aggregates, including evidence
for the mixed-valence iron oxide magnetite, are associated with amyloid plaques. To test the hypothesis
that increased chemical reduction of iron, as observed in vitro in the presence of aggregating amyloid-β,
may occur at sites of amyloid plaque formation in the human brain, the nanoscale distribution and
physicochemical states of biometals, particularly iron, were characterised in isolated amyloid plaque cores
from human Alzheimer’s disease cases using synchrotron X-ray spectromicroscopy. In situ X-ray magnetic
circular dichroism revealed the presence of magnetite: a finding supported by ptychographic observation
of an iron oxide crystal with the morphology of biogenic magnetite. The exceptional sensitivity and
specificity of X-ray spectromicroscopy, combining chemical and magnetic probes, allowed enhanced
differentiation of the iron oxides phases present. This facilitated the discovery and speciation of ferrousrich
phases and lower oxidation state phases resembling zero-valent iron as well as magnetite.
Sequestered calcium was discovered in two distinct mineral forms suggesting a dynamic process of
amyloid plaque calcification in vivo. The range of iron oxidation states present and the direct observation
of biogenic magnetite provide unparalleled support for the hypothesis that chemical reduction of iron
arises in conjunction with the formation of amyloid plaques. These new findings raise challenging questions
about the relative impacts of amyloid-β aggregation, plaque formation, and disrupted metal homeostasis
on the oxidative burden observed in Alzheimer’s disease.
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Apr 2018
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Abstract: A signature characteristic of Alzheimer’s disease (AD) is aggregation of amyloid-beta (Ab) fibrils in the brain. Nevertheless, the links between Ab and AD pathology remain incompletely understood. It has been proposed that neurotoxicity arising from aggregation of the Ab1-42 peptide can in part be explained by metal ion binding interactions. Using advanced X-ray microscopy techniques at submicron resolution, we investigated relationships between iron biochemistry and AD pathology in intact cortex from an established mouse model over-producing Ab. We found a direct correlation of amyloid plaque morphology with iron, and evidence for the formation of an iron-amyloid complex. We also show that iron biomineral deposits in the cortical tissue contain the mineral magnetite, and provide evidence that Ab-induced chemical reduction of iron could occur in vivo. Our observations point to the specific role of iron in amyloid deposition and AD pathology, and may impact development of iron-modifying therapeutics for AD.
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Sep 2017
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I10-Beamline for Advanced Dichroism
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Abstract: Iron is vital for healthy brain function. However when present in a redox-active form or in excess concentrations it can be toxic. Interestingly, increased levels of redox-active iron biominerals have been shown to exist in Alzheimer’s disease (AD) tissues, including lesions comprised of the AD peptide β-amyloid (Aβ). These iron phases are capable of producing reactive oxygen species, resulting in the generation of oxidative stress manifesting as neuronal injury. As oxidative stress and the accumulation of iron are recognised as early stage events in AD, the presence of redox-active iron may prove fundamental in the development of AD pathology. The origin of these redox-active iron biominerals is unclear but recent studies suggest their formation may involve the interaction of Aβ with unbound brain iron and/or the malfunction of the iron storage protein ferritin.
Despite these observations, the relationship between Aβ and iron is poorly understood, and the products of Aβ/iron interaction remain unknown. In this thesis, synchrotron-based x-ray techniques are combined with traditional biological approaches to examine the interactions between Aβ and various synthetic and naturally occurring iron forms. Through this methodology Aβ is shown to incorporate ferric iron phases into its fibrillar structure in vitro, with this interaction resulting in the chemical reduction of iron into a redox-active state. Further to this, Aβ is demonstrated to disrupt ferritin structure resulting in the chemical reduction of its redox-inactive iron core in vitro. Additionally the interaction of Aβ with crystalline iron phases is shown destroy iron crystal structure. Finally, redox-active iron is shown to be associated with regions of AD pathology, including fibrillar Aβ-like structures, within a transgenic mouse model of AD in situ. These findings suggest an origin for the redox-active iron forms and oxidative stress previously witnessed in AD tissue, thereby shedding light on the process of AD pathogenesis.
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Mar 2015
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I10-Beamline for Advanced Dichroism
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Open Access
Abstract: For decades, a link between increased levels of iron and areas of Alzheimer's disease (AD) pathology has been recognized, including AD lesions comprised of the peptide β-amyloid (Aβ). Despite many observations of this association, the relationship between Aβ and iron is poorly understood. Using X-ray microspectroscopy, X-ray absorption spectroscopy, electron microscopy and spectrophotometric iron(II) quantification techniques, we examine the interaction between Aβ(1–42) and synthetic iron(III), reminiscent of ferric iron stores in the brain. We report Aβ to be capable of accumulating iron(III) within amyloid aggregates, with this process resulting in Aβ-mediated reduction of iron(III) to a redox-active iron(II) phase. Additionally, we show that the presence of aluminium increases the reductive capacity of Aβ, enabling the redox cycling of the iron. These results demonstrate the ability of Aβ to accumulate iron, offering an explanation for previously observed local increases in iron concentration associated with AD lesions. Furthermore, the ability of iron to form redox-active iron phases from ferric precursors provides an origin both for the redox-active iron previously witnessed in AD tissue, and the increased levels of oxidative stress characteristic of AD. These interactions between Aβ and iron deliver valuable insights into the process of AD progression, which may ultimately provide targets for disease therapies
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Mar 2014
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