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Discovering the nanoscale iron and calcium compounds that form in Alzheimer’s disease senile plaques

Related publication: Everett J., Collingwood J. F., Tjendana-Tjhin V., Brooks J., Lermyte F., Plascencia-Villa G., Hands-Portman I., Dobson J., Perry G. & Telling N. D. Nanoscale synchrotron X-ray speciation of iron and calcium compounds in amyloid plaque cores from Alzheimer’s disease subjects. Nanoscale 10, 11782–11796 (2018). DOI: 10.1039/c7nr06794a

Publication keywords: Spectromicroscopy; STXM; Alzheimer’s disease; Senile plaques; Amyloid; Iron; Calcium

Alzheimer’s disease is the most common form of dementia, yet its cause is unclear and there is no effective treatment. A hallmark of Alzheimer’s disease is the formation of amyloid plaques in the brain that disrupt its normal function. There is also an imbalance of metals, with increased levels of iron and harmful reactive iron forms being associated with amyloid plaques.

Researchers wanted to examine Alzheimer’s disease plaques to see if there was evidence of the protein-derived amyloid peptide converting non-reactive iron forms into reactive states. They also wanted to investigate the distribution of calcium in the plaques, as disruptions to brain functions associated with calcium have also been reported in Alzheimer’s disease.

They used X-ray spectromicroscopy on the Scanning X-ray Microscopy beamline (I08) to characterise the precise distribution and chemical state of iron and calcium compounds within amyloid plaques derived from the brains of Alzheimer’s patients. They needed to examine the plaques with a resolution of tens of nanometres, whilst simultaneously accessing superb chemical sensitivity, to identify differences in metal chemistry within highly localised regions of interest. They also used X-ray Magnetic Circular Dichroism (XMCD) to probe the magnetic states of the forms of iron present within the amyloid plaques. This level of characterisation of inorganic compounds associated with amyloid plaques has not been possible in the past, and was here enabled by development and establishment of these synchrotron microscopy techniques with nanoscale resolution.

They observed iron in multiple different states within the amyloid plaque, with calcium in at least two different forms. Their results advance prior work by defining the precise properties of iron and calcium in the plaques, and show that reactive iron might serve as a target for therapies. The presence of magnetic iron forms and calcium inclusions also supports developments in non-invasive diagnosis of Alzheimer’s disease using innovative and existing clinical techniques such as Magnetic Resonance Imaging (MRI).

This study reveals, in unprecedented detail at nanoscale resolution, the properties of iron and calcium compounds in senile plaques from individuals who had Alzheimer’s disease. The findings extend current hypotheses about the way in which these and other metallic species may contribute to the pathogenesis of Alzheimer’s disease and could help direct future innovative diagnosis and treatment of the disease.

Alzheimer’s disease (AD) is the most common form of dementia, affecting ~850,000 people in the UK, with the number of cases on the increase it creates tremendous social and economic costs, and there is no cure at present.

AD is traditionally characterised by the presence of two hallmark lesions of abnormal protein aggregates: (i) intracellular neurofibrillary tangles of tau protein, and (ii) extracellular amyloid plaques, primarily comprised of amyloid-β peptides, which are small hydrophobic protein fragments (39 to 42 residues) from the transmembrane protein APP. The accumulation of amyloid plaques in AD brain tissue is thought to be fundamental to the development of the disease, affecting normal neuronal activities with intra and extracellular accumulations associated with impaired cognitive and memory function.

Alongside amyloid plaque formation, disrupted metal homeostasis is frequently observed in AD brains. For example, disrupted calcium-dependent processes have been observed in AD. Iron levels are significantly increased in several regions of AD-affected brains when compared to disease-free age-matched controls¹ and over time these chemically-reduced forms of iron, including mixed-valence iron oxides², are associated with pathological features of the disease. Iron is vital for brain function, but if it is not properly transported or stored in cells and tissues, it can catalyse the formation of free radicals. Chemically-reduced iron can produce excessive levels of these free radical species, contributing to the damage observed in AD via elevated oxidative stress and triggering of cell death processes.

This study examined the distribution and material properties of iron and calcium within amyloid plaques extracted from the brains of two AD patients. The work was underpinned by the hypothesis that the chemical reduction of iron occurs when co-localised to aggregating amyloid-β in vivo, a hypothesis supported by prior-published in vitro experiments showing amyloid-β induces the chemical reduction of ferric (Fe³⁺) iron to ferrous (Fe²⁺) states³.

With ethical approval, amyloid plaque cores were extracted and isolated from two deceased patients who had a formal diagnosis of AD. Plaques were embedded and sectioned before being examined using X-ray spectromicroscopy in the form of Scanning Transmission X-ray Microscopy (STXM), at beamline I08, and the Advanced Light Source beamline 11.0.2 (ALS; Berkeley USA). No dyes, contrast agents or aldehyde fixatives were used, which is critical as these could alter or contribute to the signal from the samples.

Nanoscale maps of the distribution of chemical species found in the plaques were created from images obtained at approximately 50 nm spatial resolution. The maps were computed from paired images, one collected at the X-ray energy corresponding to the peak absorption for a material of interest (e.g. Fe³⁺ L₃-edge absorption at 709.5 eV), and a second image collected over the same area but at a slightly lower energy (the off-peak image). The off-peak image was subtracted from the peak image, providing an artefact-free map of the distribution of the material of interest. These speciation maps were calculated using data collected at X-ray absorption edges for carbon, calcium, oxygen, and iron.

Once the overall distributions had been determined, detailed information was obtained from each area of interest by collecting a series of images (a ‘stack’) at multiple energies through an X-ray absorption edge, not just the peak/off-peak pair. For example, image stacks were acquired over the carbon K-edge (280-320 eV) and iron L_2,3-edge (700-740 eV), followed by iron L₃-edge (700-718 eV) X-ray Magnetic Circular Dichroism (XMCD) spectra revealing the magnetic properties of iron-rich regions from paired stacks obtained with left- and then right-circularly-polarised X-rays. This powerful method of spectromicroscopy produces stack images where every pixel contains detailed information about chemical and magnetic properties. In this example, the spectromicroscopy enabled detailed characterisation of individual iron deposits within the plaques.

I08 and Advanced Light Source (ALS) 11.0.2 were vital to this study for several reasons. To obtain spectromicroscopy at a length scale relevant to structures within amyloid plaque cores, spatial resolution of tens of nanometres was essential. The soft X-ray operational energy range of the beamlines enabled both organic and bioinorganic metal components of the plaques to be determined. The chemical sensitivity of these beamlines was vital in distinguishing closely-related iron species at the nanoscale, and the pioneering application of nanoscale XMCD analysis to these plaques was enabled by senior beamline scientist Dr Tohru Araki at beamline I08.

: Figure 1: STXM image (a),
speciation maps (b-f) and
composite image (g) of amyloid
plaque core material from an AD
subject. (h) Iron L2,3
-edge X-ray
absorption spectra from the
areas highlighted in (c), showing
the presence of chemically-reduced
iron. (i) Iron L3-
edge
X-ray absorption spectra and
corresponding XMCD spectra (j)
from amyloid plaque core iron
deposits, compared to a magnetite
standard (Fe3O4 ). (k) Ptychography
710 eV image (top) and iron
L3-edge speciation map (bottom)
from an amyloid plaque core iron
oxide inclusion. (l) Research team
members watch a fragile sample
being loaded into the I08 STXM.

STXM analysis at the iron L_2,3-edge revealed multiple iron phases in the amyloid plaque cores ranging from ferric, mixed valence (Fe²⁺/Fe³⁺), to ferrous and even zero-valent (Fe⁰) iron (Fig. 1h). The presence of low-oxidation-state iron (<3⁺) suggests that excessive chemical reduction of iron occurred at the amyloid plaque sites. Extended damage from free radicals has been reported at sites of amyloid aggregation, so amyloid-associated toxicity to neurons may arise from this chemical reduction of iron. XMCD spectra (Fig. 1j) revealed the presence of the magnetic mixed-valence iron phase magnetite within the plaque cores⁴. Advanced imaging with ptychography, performed at ALS beamline 11.0.2, generated images at 2 nm resolution (Fig. 1k), revealing the morphology of a maghemite-like inclusion consistent with crystalline iron of biogenic origin (as opposed to magnetite derived from pollutants of industrial origin⁵.

Calcium L-edge speciation mapping (Fig. 1d) showed evidence for at least two distinct calcium phases in the amyloid plaques, new observations that might be associated with known disruption to neuronal calcium regulation and signalling in AD.

Information was also collected regarding the organic components of the plaques by examining protein content at the carbon K-edge (Fig. 1b) and oxygen K-edge. This allowed the distribution of metals within the protein-rich organic materials of the amyloid plaque cores to be visualised for the first time at nanoscale resolution without the need to introduce any kind of labelling such as staining agents.

Conclusion: These findings associate atypical iron reduction with amyloid plaque formation, and raise challenging questions about the critical contribution of disrupted metal homeostasis to AD pathogenesis. The next step is to extend this analysis to intact sections of human brain tissue, and to characterise a wider range of the metal elements that are present. These results may prove important for clinical trials currently underway elsewhere with Alzheimer’s patients that are seeking to determine if iron-modifying drugs alter compartmental iron levels, markers of iron-associated toxicity in the brain, or clinical measures of disease progression.

References:

Tao Y. et al. Perturbed Iron Distribution in Alzheimer’s Disease Serum, Cerebrospinal Fluid, and Selected Brain Regions: A Systematic Review and Meta-Analysis. Journal of Alzheimer’s Disease 42, 679-690 (2014). DOI: 10.3233/JAD-140396
Plascencia-Villa G. et al. High-resolution analytical imaging and electron holography of magnetite particles in amyloid cores of Alzheimer’s disease. Sci. Rep. 6, 8868 (2016). DOI: 10.1038/srep24873
Everett J. et al. Ferrous iron formation following the co-aggregation of ferric iron and the Alzheimer’s disease peptide β-amyloid (1-42). J. R. Soc. Interface 11, 20140165 (2014). DOI: 10.1098/rsif.2014.0165
Collingwood J. F. et al. Iron Oxides in the Human Brain. Iron Oxides: From Nature to Applications 143–176 (John Wiley & Sons, Ltd, 2016). DOI: 10.1002/9783527691395.ch7
Allsop D. et al. Magnetite pollution nanoparticles in the human brain. Proc. Natl. Acad. Sci. 113, 10797–10801 (2016). DOI: 10.1073/pnas.1605941113

Funding acknowledgement:

This work was supported by: EPSRC grants EP/K035193/1 (JFC), EP/ N033191/1-EP/N033140/1 (JFC-NDT); the Alzheimer’s Association (AARFD-17-529742); University of Warwick alumni donations (VTT, JE); the RCMI Program from NIH at UTSA (5G12RR013646, G12MD007591), San Antonio Life Sciences Institute (SALSI)-Clusters in Research Excellence Program, Semmes Foundation; The Advanced Light Source, supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

Corresponding authors:

Dr James Everett, Keele University, [email protected] and Dr Joanna Collingwood, University of Warwick, [email protected]

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Discovering the nanoscale iron and calcium compounds that form in Alzheimer’s disease senile plaques