SCIENCE ADVANCES | RESEARCH ARTICLE

APPLIED SCIENCES AND ENGINEERING Copyright © 2021

The Authors, some
Biogenic metallic elements in the human brain? rights reserved;
exclusive licensee
American Association
James Everett1,2, Frederik Lermyte2,3, Jake Brooks2, Vindy Tjendana-Tjhin2, Germán Plascencia-Villa4, for the Advancement
Ian Hands-Portman5, Jane M. Donnelly2, Kharmen Billimoria2,6,7, George Perry4, Xiongwei Zhu8, of Science. No claim to
Peter J. Sadler6, Peter B. O’Connor6, Joanna F. Collingwood2, Neil D. Telling1* original U.S. Government
Works. Distributed
The chemistry of copper and iron plays a critical role in normal brain function. A variety of enzymes and proteins under a Creative
containing positively charged Cu+, Cu2+, Fe2+, and Fe3+ control key processes, catalyzing oxidative metabolism Commons Attribution
and neurotransmitter and neuropeptide production. Here, we report the discovery of elemental (zero–oxidation NonCommercial
state) metallic Cu0 accompanying ferromagnetic elemental Fe0 in the human brain. These nanoscale biometal License 4.0 (CC BY-NC).
deposits were identified within amyloid plaque cores isolated from Alzheimer’s disease subjects, using synchro-
tron 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.

INTRODUCTION Despite the discovery of nanoparticulate biogenic magnetite in

The transition metals iron and copper are essential for all organisms the human brain almost 30 years ago (14), information on iron and

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that undertake oxidative metabolism (1, 2) and, along with zinc, copper biomineralization at physiologically relevant spatial scales in
represent the three most abundant trace metals in the human brain the brain remains scarce, with observations being limited to micro­
(3). The ability of copper and iron to change oxidation state is cru- organisms, viruses, and plants [reviewed by Huang et al. (17)].
cial for their biological functions (1, 4, 5). They act as important These processes include the formation of elemental metallic (zero–
components of numerous enzymes that partake in redox reactions valent/oxidation state) nanoparticles from more oxidized precur-
(1, 5, 6). While copper and iron are systemically used throughout sors [e.g., (18–21)], which are much more reactive than their oxide
the human body, with Cu and Fe proteins accounting for ca. 3% of counterparts (22). This enhanced reactivity has been exploited in a
the human proteome (7), the high energy demand of the brain has range of applications, from catalysis to environmental remediation
resulted in both of these elements being extensively used there as well as antimicrobial activity (16, 23–25).
during multiple physiological processes (5, 6). Disruptions to brain metal homeostasis have been implicated in
Redox-active, catalytic copper and iron can generate toxic effects the development of multiple neurodegenerative disorders including
when they are incorrectly handled in the brain. The activity of avail- Alzheimer’s disease (AD) (26–28), the most prevalent form of de-
able, “labile” copper and iron in Fenton-like redox chemistry results mentia worldwide, currently afflicting over 25 million people (29–31).
in the production of reactive oxygen species (ROS) such as hydroxyl More specifically, altered copper and iron homeostasis has been
radicals, capable of inducing oxidative stress and cell death (8–10). linked to the formation of amyloid plaques (32–36), a hallmark patho-
The chemically reduced Cu+ and Fe2+ can catalyze these reactions, logical lesion of AD believed to be fundamentally involved in disease
further driving ROS production (10). Hence, copper and iron ho- pathogenesis (29, 37, 38). Amyloid plaques from the AD brain con-
meostasis are tightly regulated in human tissues (5, 6, 11, 12). tain chemically reduced iron oxide phases that are potentially neuro-
Previous investigations into metal neurochemistry have shown toxic and may contribute to disease progression (15, 39).
that iron oxides exist within human brain tissues (5, 13–15). Nota- Here, we use synchrotron-based scanning transmission x-ray
ble is the discovery by Kirschvink et al. (14) of single-domain nano- microscopy (STXM; see fig. S1 for microscope schematic) to deter-
crystals of the chemically reduced, magnetic iron mineral magnetite mine the nanoscale spatial distribution and chemical state of copper
extracted from postmortem brain tissue. Subsequent studies have and iron within human amyloid plaque cores from two AD sub-
revealed in situ evidence for this mineral within tissues (16). Fur- jects. STXM can generate chemically specific images to ca. 20-nm
ther understanding of metal homeostasis in the brain, and, in par- spatial resolution while simultaneously providing detailed x-ray
ticular, the role of specific oxidation states in biomineralization, absorption spectra, allowing the precise chemical state of a sample
requires chemically sensitive measurements at nanoscale resolution. to be assigned as a function of two-dimensional space. This tech-
nique requires no staining of the sample with either dyes or contrast
agents used in conventional imaging techniques, while careful con-
1
School of Pharmacy and Bioengineering, Guy Hilton Research Centre, Thornburrow trol of x-ray exposure conditions ensures that the biochemistry of
Drive, Keele University, Staffordshire ST4 7QB, UK. 2School of Engineering, Library the samples is not disturbed (15, 39, 40). In addition, using circular-
Road, University of Warwick, Coventry CV4 7AL, UK. 3Department of Chemistry, ly polarized x-rays, it is possible to correlate the magnetic properties
Technical University of Darmstadt, Alarich-Weiss-Strasse 4, 64287 Darmstadt, Germany.
4
Department of Biology and Neurosciences Institute, The University of Texas at San
of regions of interest to their specific chemical speciation via the
Antonio (UTSA), San Antonio, TX 78249, USA. 5School of Life Sciences, Gibbet Hill x-ray magnetic circular dichroism (XMCD) effect. Using these ap-
Campus, University of Warwick, Coventry CV4 7AL, UK. 6Department of Chemistry, proaches, we found nanoscale deposits of elemental metallic copper
Library Road, University of Warwick, Coventry CV4 7AL, UK. 7LGC Ltd., Queens (Cu0) and magnetic elemental iron (Fe0) within human brain tissue.
Road, Teddington TW11 0LY, UK. 8Department of Pathology, Case Western Reserve
University, Cleveland, OH 44106, USA. To the best of our knowledge, this represents a first discovery of Cu0
*Corresponding author. Email: n.d.telling@keele.ac.uk within human tissue. This discovery raises intriguing new questions

Everett et al., Sci. Adv. 2021; 7 : eabf6707 9 June 2021 1 of 10

SCIENCE ADVANCES | RESEARCH ARTICLE

about the production and role of metal nanoparticles in the brain

and whether their formation is linked to neuropathological processes.

RESULTS AND DISCUSSION

Amyloid plaque cores were isolated from the gray matter of the
frontal and temporal lobes of two (Braak stage VI) AD brains. The
full methodology used to obtain, isolate, and identify amyloid
plaque structures is in Materials and Methods. Isolated amyloid
plaques were embedded in a STXM-compatible resin and sec-
tioned to either 500 or 200 nm thickness for STXM analysis.
STXM analysis of three resin-embedded amyloid plaque cores
from the two different AD subjects are shown below. For analysis,
we adopted a multimodal x-ray absorption–based approach, de-
signed to exploit the nanoscale chemical sensitivity of STXM. Single-­
energy x-ray images were used to locate amyloid plaques and
establish overall plaque morphology. X-ray metal maps allowed the
identification of the metal, its oxidation state, and its distribution
within the amyloid plaque. Metal oxidation state difference maps
provided a qualitative distribution of differing metal oxidation states,
showing (nanoscale) variations in metal chemistry. Magnetically

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sensitive XMCD maps were used to identify strongly magnetic metal
deposits. X-ray absorption spectromicroscopy was then used to de-
termine the precise chemical state of individual metal deposits
through the generation of location-dependent x-ray absorption
spectra. This included magnetic characterization of metal deposits
via XMCD x-ray spectromicroscopy. Full details on the acquisition
of x-ray images, metal maps, and absorption spectra are in Materials
and Methods and figs. S2 to S4.

Discovery of Cu0 in subjects also displaying evidence

of chemically reduced iron
X-ray images, maps, and absorption spectra of an amyloid plaque
core from subject X are shown in Fig. 1. The x-ray image in Fig. 1A
shows the overall morphology of the plaque. The composite image
in Fig. 1B shows the plaque copper and iron content. Copper maps
Fig. 1. STXM images, metal maps, and iron and copper L2,3-edge x-ray absorp-
(see also fig. S2) showed this plaque to contain multiple discrete
tion spectra of an amyloid plaque core from subject X. (A) STXM image showing
deposits of copper in nanoscale deposits of both Cu2+ (Fig. 1B,
the overall plaque morphology. (B) Composite STXM image showing plaque mor-
green) and Cu+/Cu0 (i.e., Cu+ and/or Cu0; Fig. 1B, red). phology (blue), Cu2+ (green), Cu+/Cu0 (red), and iron (gray) content. (C) Iron oxida-
Iron mapping showed that the amyloid plaque also contains nu- tion state difference map of the region highlighted in (B). Strongly absorbing
merous nanoscale iron deposits (Fig. 1B, gray). High-resolution oxidized iron (Fe3+) is shown as light contrast, and chemically reduced iron (Fe2+
iron oxidation state difference mapping (Fig. 1C) shows a nanoscale and/or Fe0) is shown as dark contrast. (D) High-resolution composite image and
variation in the oxidation state of amyloid plaque iron. The specific (E) copper oxidation state difference map of the region highlighted in (B). In the
oxidation states of the iron deposits were determined from iron oxidation state difference map, oxidized copper (Cu2+) is shown as light contrast,
L2,3-edge x-ray absorption spectra. and chemically reduced copper (Cu+ and/or Cu0) is shown as dark contrast. (F) Iron
Differing iron phases display markedly different spectral fea- x-ray absorption spectra from the regions highlighted in (C). (G) Copper x-ray ab-
sorption spectra from the regions highlighted in (E). Energies corresponding to
tures, as illustrated in the reference spectra in Fig. 1H. Specifically,
Cu2+ and Cu+/Cu0 content are shown as dashed and dotted-dashed lines, respec-
ferric (Fe3+) materials display principal x-ray absorption features at
tively. (H and I) Reference x-ray absorption spectra from iron and copper standards
709.5 and 723 eV, whereas ferrous (Fe2+) and zero–oxidation state of varying oxidation states. (H) Iron L2,3-edge and (I) copper L2,3-edge. The energies
metallic (Fe0) phases display features at 708 and 721 eV (41). Al- corresponding to the oxidized and reduced states of the metals are shown by the
though Fe2+ and Fe0 share absorption features at identical energies, dotted-dashed and dashed lines, respectively. Copper x-ray absorption spectra.
Fe0 can be further distinguished from Fe2+ by its broadened absorp- Reprinted from Jiang et al. (42) with the permission of AIP Publishing.
tion peaks and enhanced L2/L3 peak ratios (41). Iron x-ray absorp-
tion spectra (colored circles) and their respective fits (black lines) oxidation state difference maps, and copper L2,3-edge x-ray absorp-
from the areas highlighted in Fig. 1C are displayed in Fig. 1F. Fitting tion spectra for the area highlighted in Fig. 1B (yellow square; bot-
showed area F1 to be composed of mixed oxidation states of Fe3+, tom of image).
Fe2+, and Fe0, while area F2 was predominantly Fe0. The resulting maps (Fig. 1, D and E) show a nanoscale (hun-
The chemical state of the plaque copper was examined in more dreds of nanometers) variation in copper speciation, with both oxi-
detail by collecting high-resolution Cu 2+ and Cu +/Cu 0 maps, dized (Cu2+) and chemically reduced (Cu+/Cu0) phases present.

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Copper x-ray absorption spectra from the areas highlighted in intensities (see also Fig. 1I) (42). This observation for subject Y is
Fig. 1E are shown in Fig. 1G. As shown in the reference spectra in supported by evidence for elemental metallic Cu0 in the EXAFS
Fig. 1I, Cu2+ materials display a sharp high-intensity peak at 931 eV data from subject X (Fig. 1, area G6).
and a further lower-intensity peak at 951 eV (42). In contrast, Cu+ Iron x-ray absorption spectra from iron-rich regions throughout
and Cu0 materials display peaks ca. 2.5 eV higher than Cu2+ at the plaque showed spatial variation in the iron oxidation state (Fig. 2F),
933.5 and 953.5 eV (42). From these known spectral features, it can with iron predominantly present as Fe3+ (F1: Fig. 2, C and F) as well
be seen that area G1 of the plaque contains a pure Cu2+ phase, as reduced forms (F2 and F3: Fig. 2, C and F). This was not an iso-
whereas areas G2 to G6 show both Cu2+ and Cu+/Cu0 features. For lated finding for this subject: fig. S6 shows an additional plaque
areas G2 to G4, the Cu2+ peak is dominant over Cu+/Cu0, whereas from subject Y exhibiting multiple inclusions of reduced iron.
in G5 they are approximately equal. In G6, the Cu0 peak becomes Copper mapping of a further amyloid plaque from subject X
the dominant feature. (Fig. 3) showed the presence of a single Cu+/Cu0 deposit (Fig. 3B).
These results demonstrate the presence of both iron and copper The copper x-ray absorption spectrum from this deposit (Fig. 3E)
in different chemically reduced states within the same amyloid displayed small absorption-edge steps and high post-edge absorp-
plaque. The presence of copper in multiple oxidation states within tion intensities representative of Cu0, but without the well-defined
an individual plaque suggests that redox cycling of copper may oc- EXAFS seen in the metallic Cu0 spectrum shown in Figs. 1I and
cur within these structures. A further amyloid plaque core from 2E. This spectrum may therefore represent a mixed Cu0/Cu+ phase
subject X was also found to contain chemically reduced copper, or a poorly crystalline Cu0 phase.
Cu+, shown in fig. S5.
The findings for subject X are supported by the analysis of an Discovery of ferromagnetic elemental iron
amyloid plaque from a second Alzheimer’s case (subject Y) shown Iron mapping of the amyloid plaque in Fig. 3 revealed multiple dis-
in Fig. 2, where copper (Fig. 2B) and iron (Fig. 2C) were both pres- crete areas of iron deposition. The strongest iron signal in the iron

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ent. Here, iron permeates the plaque and copper is confined to a map(s) (Fig. 3C, high-resolution map in Fig. 3F) correlated with the
single detectable Cu+/Cu0 deposit. The copper x-ray absorption region of dark contrast in the XMCD map (Fig. 3G), suggesting that
spectrum from this deposit (Fig. 2E; cyan trace) strongly resembles this iron inclusion is magnetic. The iron x-ray absorption spectrum
an elemental metallic Cu0 reference film (Fig. 2E; black trace) (42) (Fig. 3H, green) from this dense iron area (H2) was composed pri-
with additional minor contributions from Cu2+. While the spectra marily of Fe0 (94%), with only a small (6%) Fe3+ contribution. In
for Cu+ and Cu0 share principal peak positions at 933.5 and 953.5 eV, contrast, for area H1, the spectrum (Fig. 3H; orange) consisted of
metallic copper (Cu0) is discernible through the presence of well-­ contributions from Fe3+ (91%) and Fe2+ (9%).
defined extended x-ray fine structure (EXAFS) at 938 and 941.8 eV Elemental iron is one of only three transition metals that display
(42, 43). Cu0 is additionally distinguishable from Cu+ by its compa- strong ferromagnetism. Therefore, to confirm the presence of ele-
rably small absorption-edge steps and high post-edge absorption mental metallic Fe0, the fundamental magnetic properties of areas

Fig. 2. STXM images, metal maps, and copper and iron x-ray absorption spectra from a subject Y amyloid plaque. (A) STXM image showing the overall plaque
morphology. (B) Cu+/Cu0 map. (C) Iron map. (D) Composite STXM image showing plaque morphology (blue), Cu+/Cu0 (red), and iron (gray) content. (E) Copper x-ray
absorption spectrum from the copper area highlighted in (B) (cyan trace) and a Cu0 reference (black trace). Reprinted from Jiang et al. (42) with the permission of AIP
Publishing. The energies corresponding to Cu2+ and Cu+/Cu0 content are shown by the dashed and dotted-dashed lines, respectively. Asterisks highlight extended x-ray
absorption fine structure (EXAFS) indicative of metallic copper (Cu0). (F) Iron x-ray absorption spectra from the areas highlighted in (C).

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Fig. 4. Schematic displaying the experimental setup for the magnetically sen-
sitive XMCD measurements. A magnet array was inserted into the rear face of the
sample holder (magnetic field lines shown in orange), placing the sample (located
on the front face of the holder) under a magnetic field of 250 mT. The incident x-ray
beam was tuned to contain either left circularly polarized (LCP) or right circularly
polarized (RCP) light. Sample regions were examined under both LCP and RCP

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Fig. 3. STXM images, metal maps, and copper and iron x-ray absorption spec- light, with differences between these two measurements representing the XMCD
tra from a subject X amyloid plaque core. (A) Overall plaque morphology. effect (see also Materials and Methods).
(B) Cu+/Cu0 map. (C) Iron map. (D) Composite STXM image showing plaque mor-
phology (blue), Cu+/Cu0 (red), and iron (gray) content. (E) Copper x-ray absorption 2.5% dichroism effect in a 500-mT field (more than twice the field
spectrum from the copper deposit highlighted in (B). The energies corresponding
strength used here) at equivalent x-ray energies (44). These obser-
to Cu2+ and Cu+/Cu0 content are shown by the dashed and dotted-dashed lines,
vations show that this amyloid plaque iron is elemental metallic
respectively. (F) High-resolution iron map and (G) iron XMCD map from the region
highlighted in yellow in (C). In the XMCD map, areas of bright and dark contrast
rather than a mineral form of iron.
represent the presence of magnetic iron. (H) Iron x-ray absorption spectra from the XMCD measurements, performed on resin-embedded air-exposed
iron deposits highlighted in (C), (F), and (G). Fe0 nanoparticle standards, yielded spectra consistent with oxida-
tion products of metallic iron, namely, the ferrimagnetic phases
magnetite and maghemite (fig. S7 and Supplementary Text). This
H1 and H2 were probed by performing magnetically sensitive observation demonstrates how readily nanoscale Fe0 particles oxi-
XMCD measurements (see Fig. 4 and Materials and Methods for dize in air. In turn, this indicates that plaque-bound iron, where it
further experimental details), in addition to usual iron mapping. has been chemically reduced to metallic Fe0, is protected by amyloid
The corresponding circular polarization–dependent x-ray ab- encapsulation from postmortem auto-oxidation. The apparent ab-
sorption spectra and accompanying XMCD spectra, along with sence of severe surface oxidation of nanoparticulate Fe0 within the
those from a pure elemental Fe0 film standard, are shown in amyloid plaques characterized here suggests that they are endoge-
Fig. 5 (A and B). XMCD measurements of the predominantly ferric nous, rather than exogenous postmortem contamination by partic-
region, H1, showed no clear evidence of dichroism (i.e., no differ- ulate matter (PM). If the living brain acquired this iron as airborne
ence between the x-ray absorption obtained using the two different PM (45), subsequent surface reduction would be necessary to ac-
x-ray polarizations), suggesting that this region is not strongly mag- count for the observed properties of the iron particles.
netic. Conversely, the circular polarization–dependent x-ray absorp- The results presented here appear to be the first to report ele-
tion and XMCD spectra obtained from region H2 showed strong mental metallic Cu0 and Fe0 in human tissue. The observation of
dichroism (~16%), resulting in a negative XMCD peak at ca. 708 eV these phases, apparently formed within amyloid plaque cores taken
and a smaller broad positive peak (<~5%) at ca. 721 eV. The shape from the gray matter of human AD subjects, will stimulate new
and intensity of this magnetic dichroism resemble the spectra of a thinking about the role of metals in human neurobiology and may
ferromagnetic metallic Fe0 film standard. Although not identical to provide new insights into the etiology of Alzheimer’s and related
the Fe0 reference film, any differences are consistent with the pres- neurodegenerative diseases.
ence of an oxidized nonmagnetic component in the amyloid plaque The nanoscale chemical and magnetic specificities of STXM
iron rather than the formation of a magnetic iron oxide mineral have allowed us to probe the biochemistry of human brain tissue on
phase such as magnetite or maghemite, which would produce a subcellular length scales. This enabled the detection of multiple dif-
more complex three-peak XMCD spectra typical for these ferrimag- ferent chemically reduced copper and iron phases within AD amy-
netic biominerals (see fig. S7 and Supplementary Text) (15, 39). The loid plaques, revealing nanoscale metallic deposits. Many studies
strong intensity of the magnetic dichroism from this amyloid have used microfocus x-ray spectroscopy to characterize metal
plaque iron also excludes the possibility of Fe-S compounds, which chemistry in human tissues (33, 46). However, although chemically
can have x-ray absorption and XMCD spectra similar in shape to sensitive, the spatial resolution of these techniques leads to spatial
metallic iron, but show a much weaker dichroism. For example, the averaging only across the micrometer scale, precluding the precise
most magnetic Fe-S mineral, pyrrhotite, was found to exhibit only a identification of the highly reduced nanoscale metal deposits observed

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Fig. 5. XMCD measurements of a subject X amyloid plaque core. (A) LCP and RCP iron x-ray absorption spectra, and (B) XMCD spectra from the amyloid plaque iron
shown in Fig. 3, and a thin Fe0 reference film.

here. The ability to separate spatially different metal phases with reduction, in some cases coupled to the oxidation of glucose (53).
nanoscale resolution using STXM techniques provides unique char- There may be analogous reductases in the human brain, including
acterization of copper and iron in tissues. cysteine-rich Zn2+- and Cu+-binding metallothionein-3, a neuropro-
There are several plausible mechanisms for the formation of ele- tective protein with thiolate clusters, capable of reducing A-Cu2+
mental metallic Cu0 and Fe0 in amyloid deposits. The first is direct to A-Cu+ (55).
reduction of A-bound Cu+/2+ and Fe2+/3+. Given the reportedly Because A-Cu2+ is readily reduced to A-Cu+ by multiple bio-
high reduction potential of A-Cu composites compared to other logical reductants (56), Cu0 nanoclusters might be formed through
biomolecule-Cu complexes, this is feasible (47). Supporting this, we the disproportionation of A-bound Cu+ into Cu2+ and Cu0. Cu+
collected in vitro mass spectrometry (MS) data with collision-­ appears to prefer a linear or trigonal binding geometry to A (e.g.,
induced dissociation (CID) (see figs. S8 to S10 and Supplementary involving His13 and His14), in which it could readily bind O2 (57).
Text), which showed that A(1–42) binds Cu2+, in the N-terminal Under conditions of hypoxia, which are related to neurodegenera-
region. Analysis of the isotope distributions of Cu-bound fragments tion and increased A accumulation (58), aggregates of A-Cu+ un-
demonstrated that Cu2+ reduction to Cu+ occurs during the CID able to bind O2 may instead destabilize and disproportionate to
process. Accompanying a- and c-type peptide fragments, formed Cu2+ and Cu0.
from hydrogen-deficient peptide cation radicals (48), were also ob- Under conditions of oxidative stress, oxidative attack on coordi-
served, providing direct evidence that A(1–42) acts as the reducing nated amino acids such as Cys or His could disrupt the Cu coordi-
agent during the conversion of Cu2+ to Cu+. Thus, the reduction of nation sphere, destabilizing Cu+ toward disproportionation. Such
copper by A(1–42) is a potential source of the chemically reduced oxidative attack is feasible in the context of the oxidative damage
Cu phases observed in AD amyloid plaque cores as evidenced observed in AD brain tissue. Possible oxidants include ROS, such as
by STXM. hydroxyl radicals (produced, for example, by an A-Cu redox cycle)
Natural hydride donors such as NAD(P)H (reduced form of (57) and hypochlorous acid, which has been associated with amy-
nicotinamide adenine dinucleotide phosphate) and formate may also loid burden in AD mouse models (59). This possibility is supported
be capable of reducing copper and iron to metallic forms. Other by the observation of oxidized Cu-binding amino acid residues
transition metals (notably, Ru, Os, and Ir) can accept hydride, al- (e.g., His6, His13, His14, and Tyr10) in A-Cu complexes in vitro
though no in vitro evidence yet exists for copper and iron. It is no- (also shown in supporting MS data here) and A plaques from the
table that enzymes containing iron-sulfur clusters use formate as an brains of AD patients (57, 60).
electron donor (49–51). A final intriguing possibility is the catechol- and phenol-oxidizing
Multiple bacteria, fungi, and plants produce elemental metallic activity of A-Cu2+. There is evidence for a dinuclear Cu2+ center in
nanoclusters, including Cu0 and Fe0, as a means of resistance to A-Cu aggregates that is capable of both monooxygenase and oxi-
metal toxicity (52–54). The mechanisms for these syntheses are not dase activity, similar to the catechol oxidase and tyrosinase enzymes
fully understood, but are thought to proceed through enzymatic (61). The proposed inactivation mechanism for tyrosinase occurs

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when oxy-tyrosinase processes a catechol as a phenol, triggering its cut into 1-cm slices, and stored at −70°C. The frozen tissue slices
monooxygenation to a hydroxyquinone rather than its oxidation to were thawed, and the gray matter from the frontal and temporal
an ortho-quinone. This mechanism results in the reductive elimina- lobes was isolated through the removal of the blood vessels, meninges,
tion of one Cu as Cu0 (62). Over time, in the presence of excess and white matter. The isolated gray matter was then homogenized
catechol, tyrosinase is gradually deactivated, as the buildup of Cu0 by heating to 95°C in the presence of 2% SDS in 50 mM tris buffer.
limits the number of catalytically active Cu2+ centers. In the case of This homogenate was filtered (100-m pore size) to remove any re-
A-Cu2+, its oxidation of catecholamines like l-DOPA and dopa- maining large tissue debris and pelleted through centrifugation at
mine (both present in high concentrations in the brain) could result 800 rpm. AD gray matter was then further homogenized through
in a similar inactivation mechanism, producing Cu0 that is then sta- the addition of 0.1% (w/v) SDS, 150 mM NaCl, and 0.02% NaN3
bilized and protected from subsequent oxidation by the A aggre- (w/v) before being filtered (35 m pore size) and pelleted at 1000 rpm
gate. The ability of A to stabilize copper in a Cu0 state would offer for ca. 30 min. Amyloid plaque cores were isolated from the filtrate
an explanation for the recently reported enrichment of isotopically through ultracentrifugation at 20,000 rpm in a sucrose gradient [1.8 to
light copper in AD brains compared to disease-free controls (63). 1.2 M sucrose, in a 0.1% (w/v) SDS, 150 mM NaCl, and 0.02% (w/v)
The accumulation of reduced metallic copper and iron observed NaN3 solution]. The resulting fractions were collected, before being
here within amyloid plaques may induce oxidative stress through recovered for a final time with 0.1% (w/v) SDS, 150 mM NaCl, and
the localized overproduction of ROS (9, 10, 64, 65), thereby contrib- 0.02% (w/v) NaN3, and concentrated via centrifugation at 1200 rpm.
uting to the pattern of neuroinflammation and neuronal failure in Forty microliters of pelleted amyloid plaque core material was
AD-affected brain areas. The presence of metallic elemental nano­ transferred into a centrifugal concentrator (Corning Spin-X UF;
particles would be expected to further exacerbate these effects due 40-kDa cutoff) and spun at 6690 rpm for 10 min. Amyloid plaque
to the increased reactivity of metallic phases compared to their cores were dehydrated through an ethanol series (100 l; 40 to 100%
oxide counterparts. Alternatively, the sequestering of redox-active dry), with waste ethanol being removed at each step through cen-

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and catalytically active metals by amyloid may represent an antiox- trifugation at 6690 rpm for 10 min. No other chemical fixatives were
idant mechanism by preventing metal-catalyzed oxidation of near- introduced to prevent metal leeching and preserve mineral compo-
by cellular structures (66). The process of amyloid plaque formation sition within the amyloid plaque cores. Following dehydration, am-
and degradation in human brains, for both healthy individuals and yloid plaque cores were embedded in an STXM-compatible resin
those with AD, remains to be fully elucidated. composed of an equimolar mixture of trimethylolpropane triglyc-
Transition metals that can be associated with specific disease pa- idyl ether: 4,4′-methylenebis (2-methylcyclohexylamine). This res-
thologies offer potential for disease diagnosis and staging, particu- in has previously been used successfully by our group to examine
larly with the application of advances in iron-sensitive clinical biological tissues using x-ray spectromicroscopy (15, 39, 68–70).
imaging (67). Chemically reduced copper and iron associated with Resin polymerization occurred at 60°C over a period of 24 hours.
amyloid structures may also represent an innovative target for alter- Semi-thin sections, 500 or 200 nm thickness, of embedded amyloid
native AD therapies intended to lower the oxidative stress burden plaque cores were cut with a Reichert-Jung Ultra-cut microtome,
in affected brain regions. While it is critical that metal-modifying using a nonmetallic (diamond) blade to minimize the risk of metal
strategies are not detrimental to essential metabolic processes de- contamination during the sectioning process. Sections containing
pendent on these elements, targeting the interaction of amyloid embedded amyloid plaque cores were deposited onto transmission
with copper and iron may alter neurotoxicity arising from this in- electron microscopy (TEM) grids (Agar Scientific; 100 mesh), and
teraction, thereby modifying disease progression. these grids were, in turn, mounted onto sample plates for STXM
The unexpected identification of Cu0 and Fe0 within AD amy- examination.
loid plaques suggests that biogenic metallic elements, previously The presence of amyloid plaque material within the sectioned
observed only in microorganisms, viruses, and plants, can also oc- sample material was confirmed via staining with 1% (w/v) Congo
cur in humans. The reactivity of these metallic phases differs from red solution and subsequent examination using cross-polarized op-
their metal oxide counterparts previously detected in the human brain tical microscopy as shown in figure 1 of Everett et al. (15). Congo
and has the scope to redefine our understanding of metal neurochem- red–stained sections were not used for STXM experiments.
istry and the role of metal toxicity in neurodegenerative diseases.
Preparation of metal standards
Copper(II) oxide (CuO) nanopowder (<50 nm particle diameter)
MATERIALS AND METHODS and Fe0 nanoparticles (25 to 45 nm diameter; both from Sigma-­
Preparation of amyloid plaque cores for STXM analysis Aldrich) were embedded in an STXM-compatible resin composed
Human brain tissue was obtained from two AD subjects at autopsy of an equimolar mixture of trimethylolpropane triglycidyl ether,
with the informed written consent of their relatives. The methodol- 4,4′-methylenebis (2-methylcyclohexylamine), as used during the
ogy used to obtain, isolate, and identify amyloid plaque structures embedding of the amyloid plaque cores. Semi-thin sections (100 to
from the brain tissue was approved by the Bioethics Committee 500 nm thickness) of the embedded metal standards were cut with a
(Department of Pathology, Case Western Reserve University), and Reichert-Jung Ultra-cut microtome using a diamond blade and were
this study was performed under U.K. ethical approval 07/MRE08/12 mounted onto TEM grids (Agar Scientific; 100 mesh) for STXM
and U.S. IRB 03-00-26. The process of amyloid plaque core prepa- examination. Both the embedding and sectioning of the metal nano­
ration for STXM analysis has also been previously described by particles were performed under ambient conditions. These stan-
Everett et al. (15). dards were prepared to assess the effect of the embedding process
The brains from the two confirmed AD subjects (Braak stage VI) and to confirm that the STXM measurements did not alter the oxi-
were removed at autopsy (5 hours postmortem), divided into half, dation state of metals embedded within an organic matrix. An

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SCIENCE ADVANCES | RESEARCH ARTICLE

additional ferric (Fe3+) iron standard was also prepared in an organic of STXM scanning parameters” section and the Supplementary
polymer matrix [as described by Brooks et al. (68)] to further assess Materials).
the effect on iron oxidation state of successive x-ray beam doses. To Scanning transmission x-ray microscopy–x-ray magnetic
avoid cross-contamination of samples, the preparation of metal circular dichroism
nanoparticle standards was performed after the preparation and To establish the magnetic state of the iron inclusions identified with-
storage of amyloid plaque core materials. in the amyloid plaque cores, magnetically sensitive XMCD measure-
ments were performed. These measurements were facilitated by
Scanning transmission x-ray microscopy inserting a NdFeB ring magnet array (allowing x-ray transmission)
STXM experiments were performed at the Advanced Light Source into the rear face of the STXM sample holder and mounting the
(Lawrence Berkeley National Laboratory, CA, USA) beamline 11.0.2 sample to the front face of the holder (see Fig. 4 for STXM-XMCD
and Diamond Light Source (Oxfordshire, UK) beamline I08. Fo- sample holder schematic and fig. S1 for STXM beamline schematic).
cused x-ray spot sizes were ≤50 nm at both sources. Single-energy The magnetic field strength at the sample position was measured to
x-ray images taken at the iron L3-edge (706 eV; fig. S6) or copper be ~250 mT, which we have previously shown is sufficient to induce
L3-edge (928 eV; Figs. 1 to 3 and figs. S2 and S5) were used to locate a significant degree of magnetic polarization in iron mineral (e.g.,
and visualize the overall morphology of the amyloid plaques. magnetite) standards, as well as magnetic iron inclusions within
STXM metal maps amyloid plaque cores (15). X-ray absorption spectra from such mag-
To generate maps showing the spatial distribution of metal phases netized samples were created by performing paired stacks over the
associated with the amyloid plaque cores, paired images were taken: iron L2,3-edge using both left and right circularly polarized (RCP)
one peak image at the energy corresponding to a feature of interest light. The dichroism is revealed as the difference spectrum obtained
(e.g., the Cu2+ L3-edge peak; 931 eV) and a further off-peak image a by subtracting the x-ray absorption spectra obtained using RCP
few electron volts below this feature. The off-peak image was then light from the equivalent spectra obtained using left circularly po-

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subtracted from the peak image, yielding a metal map. This method larized (LCP) light (see, e.g., fig. S7).
of metal mapping, shown in fig. S2, allows artifacts from the embed- To visualize iron deposits showing strong magnetic polarization
ding resin to be removed, providing a true representation of the (i.e., strongly magnetic deposits), XMCD maps were also created.
metal distribution within the amyloid plaque cores. By finely tuning These were obtained by first averaging five images taken close to the
the energy of the incident x-ray beam, metal distribution maps Fe2+/Fe0 L3-edge peak energy position for both LCP and RCP mea-
showing distinct oxidation states were generated by exploiting the surements. The averaged RCP image was then subtracted from the
preferential absorption of x-rays of differing energies by specific equivalent LCP image to yield an XMCD difference map, with areas
metal phases (e.g., Cu2+ at 931 eV and Cu+/Cu0 at 933.5 eV). Metal of bright or dark contrast indicating significant dichroism effects
maps were created at the iron L3-edge (710 eV) showing iron distri- (e.g., Fig. 3G).
bution and two energies at the copper L3-edge (ca. 931 and 933.5 eV) Optimization of STXM scanning parameters
showing Cu2+ and Cu+/Cu0 content, respectively. To best preserve the native chemistry of the amyloid plaque copper
Additional iron and copper oxidation state difference maps were and iron upon examination with STXM, control measurements were
created by subtracting images taken at energies corresponding to a first performed on ferric (Fe3+) and cupric (Cu2+) metal oxide stan-
chemically reduced state (Fe2+/Fe0 [708 eV], Cu+/Cu0 [933.5 eV]) dards to assess the effect of x-ray beam dose on the oxidation state
from the oxidized state (Fe3+ [709.5 eV], Cu2+ [931 eV]). The result- of these metals.
ing maps provide a qualitative distribution of the different iron and The purpose of these control measurements was to ascertain
copper oxidation states within the plaque, with reduced phases STXM scan parameters that generate sufficient signal:noise to de-
showing as dark contrast and oxidized phases showing as light con- termine metal oxidation state while simultaneously limiting x-ray
trast (see also fig. S3). doses to ensure negligible x-ray beam–induced changes to sample
Metal x-ray absorption spectra chemistry, thereby validating the use of these methodologies for the
X-ray absorption spectra providing detailed information regarding examination of metal oxidation state. This is typically achieved by
the oxidation state of metal phases within the amyloid plaque cores tuning the incident beam intensity by, for example, increasing/
were obtained from a series of images (called a “stack”) taken over a decreasing exposure (dwell) times or altering the beamline optics
desired energy absorption edge (see fig. S4). Raw x-ray absorption such as the exit slits. Because of the inherent variations in synchro-
intensities were normalized to the incident x-ray beam by conver- tron light source specifications, the x-ray dose tolerance of sample
sion to optical density using background regions that did not materials, and the scientific questions under investigation, these pa-
contain any sample material. Using this approach, specific x-ray rameters must be empirically tailored for each individual experi-
absorption spectra can be generated from each pixel of an image, ment. The experiments described below demonstrate our method
allowing the chemical composition of highly localized (<50 nm in for optimizing the specific STXM scanning parameters used in
this instance) regions of interest to be determined. Stacks were ac- this study.
quired over the iron L2,3-edge (700 to 740 eV) and copper L2,3-edge To determine the optimal scan parameters for iron L-edge spec-
(920 to 960 eV). For stack measurements, the dark count (back- tromicroscopy, we performed a series of repeat iron L3-edge STXM
ground noise attributable to the beamline) was subtracted before measurements on a ferric standard within an organic polymer ma-
the generation of the x-ray absorption spectra. When examining trix, using differing x-ray doses controlled by altering the beamline
amyloid plaque material, dwell (exposure) times per pixel were exit slit size. We determined an appropriate x-ray dose to be where
kept as short as possible while ensuring sufficient signal:noise to no changes in the iron L3-edge spectra were observed over succes-
minimize photon-dose effects, thereby negating any substantial sive iron L3-edge measurements. Scans were performed with either
alteration of native sample chemistry (see also the “Optimization 10- or 5-m exit slit sizes. No perceivable changes to Fe3+ oxidation

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SCIENCE ADVANCES | RESEARCH ARTICLE

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SCIENCE ADVANCES | RESEARCH ARTICLE

79. C. Hureau, Coordination of redox active metal ions to the amyloid precursor protein Fulbright-Warwick Scholarship (J.M.D.), Kleberg Foundation 2019 Medical Research Grant,
and to amyloid- peptides involved in Alzheimer disease. Part 1: An overview. Coord. Kleberg Foundation 2020 Medical Research Grant, NIH NIA R01AG066749, Alzheimer’s
Chem. Rev. 256, 2164–2174 (2012). Association grant AARFD-17-529742 (G.P.-V.), The Lowe Foundation, and Semmes Foundation.
Author contributions: Conceptualization: J.E., F.L., G.P., P.J.S., P.B.O., J.F.C., and N.D.T.
Acknowledgments: We thank S. Siedlak (Case Western Reserve University, Cleveland OH, Methodology: J.E., F.L., G.P.-V., G.P., J.F.C., and N.D.T. Investigation: J.E., F.L., J.B., V.T.-T., G.P.-V.,
USA) for technical assistance in isolation of the amyloid plaque cores. We thank B. Kaulich, I.H.-P., J.M.D., K.B., G.P., J.F.C., and N.D.T. Visualization: J.E., F.L., G.P.-V., G.P., P.J.S., P.B.O., J.F.C.,
T. Araki, and M. Kazemian for their support at Diamond Light Source beamline I08, and D. Vine and N.D.T. Supervision: J.F.C. and N.D.T. Writing—original draft: J.E. Writing—review and
for support at the Advanced Light Source beamline 11.0.2. The amyloid plaque cores were editing: J.E., F.L., J.B., V.T.-T., G.P.-V., I.H.-P., J.M.D., K.B., G.P., X.Z., P.J.S., P.B.O., J.F.C., and N.D.T.
isolated from donated tissues obtained with informed consent and were analyzed in Competing interests: The authors declare that they have no competing interests. Data and
accordance with the Declaration of Helsinki under the remit of ethical approval 07/MRE08/12 materials availability: All data needed to evaluate the conclusions in the paper are present in
from the UK National Research Ethics Service. The amyloid plaque cores in this study were the paper and/or the Supplementary Materials. The datasets from this study are available
analyzed under the remit of a material transfer agreement between the University of Texas at through the Keele University research repository at: http://doi.org/10.21252/g99p-0058.
San Antonio and the University of Warwick. The Advanced Light Source is supported by the
Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy Submitted 11 November 2020
under contract no. DE-AC02-05CH11231. Funding: This work was supported by the following: Accepted 22 April 2021
Engineering and Physical Sciences Research Council grant EP/K035193/1 (J.F.C.), Engineering and Published 9 June 2021
Physical Sciences Research Council grant EP/N033191/1 (J.F.C., P.B.O., and P.J.S.), Engineering and 10.1126/sciadv.abf6707
Physical Sciences Research Council grant EP/N033140/1 (N.D.T.), Engineering and Physical
Sciences Research Council grant EP/P030572/1 (P.J.S.), Engineering and Physical Sciences Citation: J. Everett, F. Lermyte, J. Brooks, V. Tjendana-Tjhin, G. Plascencia-Villa, I. Hands-Portman,
Research Council grant EP/N509796/1 (J.B.), Engineering and Physical Sciences Research J. M. Donnelly, K. Billimoria, G. Perry, X. Zhu, P. J. Sadler, P. B. O’Connor, J. F. Collingwood, N. D. Telling,
Council grant EP/L015307/1 (K.B.), University of Warwick alumni donations (J.E. and V.T.-T.), Biogenic metallic elements in the human brain?. Sci. Adv. 7, eabf6707 (2021).

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