I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[29042, 24534]
Open Access
Abstract: Neuromelanin-pigmented neurons of the substantia nigra are selectively lost during the progression of Parkinson’s disease. These neurons accumulate iron in the disease state, and iron-mediated neuron damage is implicated in cell death. Animal models of Parkinson’s have evidenced iron loading inside the nucleoli of nigral neurons, however the nature of intranuclear iron deposition in the melanised neurons of the human substantia nigra is not understood. Here, scanning transmission x-ray microscopy (STXM) is used to probe iron foci in relation to the surrounding ultrastructure in melanised neurons of human substantia nigra from a confirmed Parkinson’s case. In addition to the expected neuromelanin-bound iron, iron deposits are also associated with the edge of the cell nucleolus. Speciation analysis confirms these deposits to be ferric (Fe3+) iron. The function of intranuclear iron in these cells remains unresolved, although both damaging and protective mechanisms are considered. This finding shows that STXM is a powerful label-free tool for the in situ, nanoscale chemical characterisation of both organic and inorganic intracellular components. Future applications are likely to shed new light on incompletely understood biochemical mechanisms, such as metal dysregulation and morphological changes to cell nucleoli, that are important in understanding the pathogenesis of Parkinson’s.
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Aug 2024
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I08-Scanning X-ray Microscopy beamline (SXM)
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James
Everett
,
Jake
Brooks
,
Vindy Tjendana
Tjhin
,
Frederik
Lermyte
,
Ian
Hands-Portman
,
Germán
Plascencia-Villa
,
George
Perry
,
Peter J.
Sadler
,
Peter B.
O’connor
,
Joanna F.
Collingwood
,
Neil D.
Telling
Open Access
Abstract: The accumulation of amyloid plaques and increased brain redox burdens are neuropathological hallmarks of Alzheimer’s disease. Altered metabolism of essential biometals is another feature of Alzheimer’s, with amyloid plaques representing sites of disturbed metal homeostasis. Despite these observations, metal-targeting disease treatments have not been therapeutically effective to date. A better understanding of amyloid plaque composition and the role of the metals associated with them is critical. To establish this knowledge, the ability to resolve chemical variations at nanometer length scales relevant to biology is essential. Here, we present a methodology for the label-free, nanoscale chemical characterization of amyloid plaques within human Alzheimer’s disease tissue using synchrotron X-ray spectromicroscopy. Our approach exploits a C–H carbon absorption feature, consistent with the presence of lipids, to visualize amyloid plaques selectively against the tissue background, allowing chemical analysis to be performed without the addition of amyloid dyes that alter the native sample chemistry. Using this approach, we show that amyloid plaques contain elevated levels of calcium, carbonates, and iron compared to the surrounding brain tissue. Chemical analysis of iron within plaques revealed the presence of chemically reduced, low-oxidation-state phases, including ferromagnetic metallic iron. The zero-oxidation state of ferromagnetic iron determines its high chemical reactivity and so may contribute to the redox burden in the Alzheimer’s brain and thus drive neurodegeneration. Ferromagnetic metallic iron has no established physiological function in the brain and may represent a target for therapies designed to lower redox burdens in Alzheimer’s disease. Additionally, ferromagnetic metallic iron has magnetic properties that are distinct from the iron oxide forms predominant in tissue, which might be exploitable for the in vivo detection of amyloid pathologies using magnetically sensitive imaging. We anticipate that this label-free X-ray imaging approach will provide further insights into the chemical composition of amyloid plaques, facilitating better understanding of how plaques influence the course of Alzheimer’s disease.
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Mar 2024
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[15230, 24534, 29042, 30776]
Open Access
Abstract: The synchrotron x-ray spectromicroscopy technique Scanning Transmission X-ray Microscopy (STXM) offers a powerful means to examine the underlying biochemistry of biological systems, owing to its combined chemical sensitivity and nanoscale spatial resolution. Here we introduce and demonstrate methodology for the use of STXM to examine the biochemistry of the human brain. We then discuss how this approach can help us better understand the biochemical changes that occur during the development of degenerative brain disorders, potentially facilitating the development of new therapies for disease diagnosis and treatment.
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Jun 2023
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I08-Scanning X-ray Microscopy beamline (SXM)
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James
Everett
,
Frederik
Lermyte
,
Jake
Brooks
,
Vindy
Tjendana-Tjhin
,
Germán
Plascencia-Villa
,
Ian
Hands-Portman
,
Jane M.
Donnelly
,
Kharmen
Billimoria
,
George
Perry
,
Xiongwei
Zhu
,
Peter J.
Sadler
,
Peter B.
O'Connor
,
Joanna F.
Collingwood
,
Neil D.
Telling
Diamond Proposal Number(s):
[15854]
Open Access
Abstract: The chemistry of copper and iron plays a critical role in normal brain function. A variety of enzymes and proteins containing positively charged Cu+, Cu2+, Fe2+, and Fe3+ control key processes, catalyzing oxidative metabolism and neurotransmitter and neuropeptide production. Here, we report the discovery of elemental (zero–oxidation state) metallic Cu0 accompanying ferromagnetic elemental Fe0 in the human brain. These nanoscale biometal deposits were identified within amyloid plaque cores isolated from Alzheimer’s disease subjects, using synchrotron x-ray spectromicroscopy. The surfaces of nanodeposits of metallic copper and iron are highly reactive, with distinctly different chemical and magnetic properties from their predominant oxide counterparts. The discovery of metals in their elemental form in the brain raises new questions regarding their generation and their role in neurochemistry, neurobiology, and the etiology of neurodegenerative disease.
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Jun 2021
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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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I06-Nanoscience (XPEEM)
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Diamond Proposal Number(s):
[838]
Abstract: Biogenic nanoscale vanadium magnetite is produced by converting V(V)- bearing ferrihydrites through reductive transformation using the metal- reducing bacterium Geobacter sulfurreducens. With increasing vanadium in the ferrihydrite, the amount of V-doped magnetite produced decreased due to V-toxicity which interrupted the reduction pathway ferrihydrite – magnetite, resulting in siderite or goethite formation. Fe L2,3 and V L2,3 X-ray absorption spectra and data from X-ray magnetic circular dichroism analysis revealed the magnetite to contain the V in the Fe(III) Oh site, predominately as V(III) but always with a component of V(VI) present a consistent V(IV)/V(III) ratio in the range 0.28 to 0.33. The bacteriogenic production of V-doped magnetite nanoparticles from V-doped ferrihydrite is confirmed and the work reveals that microbial reduction of contaminant V(V) to V(III)/V(IV) in the environment will occur below the Fe-redox boundary where it will be immobilised in biomagnetite nanoparticles.
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Jul 2020
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I08-Scanning X-ray Microscopy beamline (SXM)
I10-Beamline for Advanced Dichroism - scattering
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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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