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Mass spectrometry imaging shows major derangements in neurogranin and in purine
metabolism in the triple-knockout 3×Tg Alzheimer mouse model.
Clara Esteve1, Emrys A. Jones1, Douglas B. Kell2,3, Hervé Boutin4,5, Liam A.
McDonnell*1,6
1Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden,
The Netherlands
2School of Chemistry, The University of Manchester, Manchester, Lancs M13 9PL, UK
3Manchester Institute of Biotechnology, The University of Manchester, 131 Princess St,
Manchester, Lancs, UK
4Faculty of Medicine and Human Sciences, The University of Manchester, Manchester,
UK
5Wolfson Molecular Imaging Center, The University of Manchester, Manchester, UK
6Fondazione Pisana per la Scienza ONLUS, Pisa, Italy.
* Corresponding author and reprint requests
Dr. Liam A. McDonnell, Center for Proteomics and Metabolomics, Leiden University
Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands; E-mail:
[email protected]; Phone: +31 71 526 8744; Fax: +31 71 526 6907
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Abstract
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI)
can simultaneously measure hundreds of biomolecules directly from tissue. Using
different sample preparation strategies, proteins and metabolites have been profiled to
study the molecular changes in a 3×Tg mouse model of Alzheimer’s disease. In
comparison with wild-type (WT) control mice MALDI-MSI revealed Alzheimer’s
disease-specific protein profiles, highlighting dramatic reductions of a protein with m/z
7560, which was assigned to neurogranin and validated by immunohistochemistry. The
analysis also revealed substantial metabolite changes, especially in metabolites related
to the purine metabolic pathway, with a shift towards an increase in
hypoxanthine/xanthine/uric acid in the 3×Tg AD mice accompanied by a decrease in
AMP and adenine. Interestingly these changes were also associated with a decrease in
ascorbic acid, consistent with oxidative stress. Furthermore, the metabolite N-
arachidonyl taurine was increased in the diseased mouse brain sections, being highly
abundant in the hippocampus. Overall, we describe an interesting shift towards pro-
inflammatory molecules (uric acid) in the purinergic pathway associated with a decrease
in anti-oxidant level (ascorbic acid). Together, these observations fit well with the
increased oxidative stress and neuroinflammation commonly observed in AD.
Keywords: Alzheimer’s, 3×Tg mouse, neurogranin, purigenic pathway, mass
spectrometry imaging
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Introduction
Alzheimer’s disease or Alzheimer-type dementia (AD) is a neurodegenerative disorder
and the most common form of dementia. Today AD is the largest unmet medical need in
neurology [1, 2]. As such, it represents a massive and growing health-care problem with
about 35 million patients in 2010, expected to reach 115 million by 2050, with 2/3 of
those patients living in low- to middle-income countries and the cost of long-term care
predicted to double over the next 50 years (from 1.2% to 2.5% of European countries’
GDP) [3-6]. To date, only symptomatic treatments are available and there is an urgent
need to investigate new approaches and concepts to develop new therapies, which
essentially depend on a much better understanding of the pathophysiology of AD. From
a physiopathological point of view, Alzheimer’s disease is characterised by several
abnormalities, such as elevated levels of amyloid- (Aβ) peptides leading to Aβ plaque
formation, neurofibrillary tangles (NFT) made of aggregates of abnormally
phosphorylated Tau proteins, alteration of the cholinergic system, brain atrophy,
decreased brain metabolism and neuroinflammation [7-9], not all of which contribute
[10] to the end point of synaptic and neuronal loss, and cognitive dysfunction. Iron
dysregulation leading to oxidative stress has also been strongly implicated in disease
progression [11-16]. Iron dysregulation may also interact with a potential microbial
component of inflammation [17-19] for which there is other and wide-ranging evidence
[20-23].
Traditionally, histopathological analysis of AD tissue relies on a priori hypotheses or
knowledge, and the use of known biomarkers and associated probes (e.g. antibodies,
radiolabelled tracers). Conversely, mass spectrometry imaging (MSI), commonly based
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on matrix-assisted laser desorption/ionization (MALDI), can be used to image hundreds
of biomolecules directly from tissue [24, 25]. A number of mass spectrometric methods
have been developed for MSI, enabling high sensitivity analysis of a range of molecular
classes, including proteins, peptides and metabolites directly from tissue [26]. This is a
data-driven strategy [27], and thus requires no a priori knowledge about hypothetical
biomarkers. MSI can be applied to different molecular classes just by altering the tissue
preparation strategy [28, 29]. In many neurological diseases the pathophysiology is not
entirely known and a beneficial approach involves systematic investigations of the
biomolecular differences between diseased and healthy tissue. MSI has been used to
investigate spatiotemporal molecular changes in several neurological pathological
conditions in rodent models, including ischemic stroke [30-32], cortical spread
depression [29], Parkinson’s disease [33, 34] and Alzheimer’s disease [35, 36]. A key
advantage of MSI is that it can annotate tissues based on their MS profiles and thereby
distinguish biomolecularly distinct regions even if they are unexpected or cannot be
distinguished using established histological and histochemical methods [37, 38]. This is
especially true for disorders such Alzheimer’s disease, which is a complex and
multifactorial disease and for which the pathophysiology is not well understood.
The triple transgenic (3Tg) mouse model [39] expressed the human mutations for
Presenilin (PresenilinM146V), the amyloid protein precursor APPSwe essential to obtain
amyloid pathology in mice [40] as well as the tauP301L mutation that induces tau
pathology [41, 42], and is considered to present all the pathological hallmarks of AD.
Here we report the results of a MALDI MSI study of the biomolecular changes related
to Alzheimer-type disease in the brain of the 3Tg mouse, including both metabolites and
proteins.
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Materials and Methods
Chemicals
9-aminoacridine (9-AA), sinapinic acid (SA), poly-L-lysine solution, isopropanol,
methanol (MeOH), ethanol and trifluoroacetic acid (TFA) were obtained from Sigma-
Aldrich (St. Louis, MO, USA). Indium tin oxide (ITO)-obtained slide glass were
purchased from Bruker Daltonics (Bremen, Germany).
Animals
Wild type (WT) and triple transgenic (3×Tg) incorporating the transgenes PS1M146V,
APPSwe, and tauP301L [39] adult male mice (n = 3 per group) were kept under a 12 h
light–dark cycle (08:00/20:00 h) at 22 °C with free access to food and water. Mice were
killed by anaesthetic overdose with isoflurane and decapitated. Brains were rapidly (<60
s) removed and frozen in cooled (-40 °C) isopentane. All procedures were carried out in
accordance with the Animals (Scientific Procedures) Act 1986.
MSI Data Acquisition
For the MALDI MSI experiments sagittal tissue sections of 12 µm thickness were cut
with a cryostat (1720 Digital, Leica, Rijswijk, The Netherlands) at -20 °C. Next these
were thaw-mounted onto an ITO-coated glass slides (Delta Technologies, Stillwater,
MN) previously coated with poly-L-lysine (0.05 % in water), and stored at -80 °C.
Before use, the tissues were slowly brought to room temperature in a desiccator.
Energy-rich metabolites, such as ATP, can degrade in tissue. It has been shown that
post-mortem degradation is more rapid under normal physiological conditions
(Gündisch et al., 2012). Note: there are technologies available specifically designed to
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limit metabolite degradation (Sugiura et al., 2014; Blatherwick et al., 2013) but they
were not available at the University of Manchester where the animal experiments were
performed. Instead, we prepared the tissues in a manner to try to limit metabolic
degradation, specifically
• All brains were excised and frozen in cooled (-40 C) isopentane within 60s of
sacrifice;
• All ITO slides were pre-cooled in the cryostat chamber prior to tissue mounting;
• All samples were mounted within the chamber and at the same time - tissues not
being cut were covered in foil to avoid drying;
• Sections were taken and placed onto the relevant slide with just enough heat
applied to the underside of the slide to adhere the tissue section, and then immediately
refrozen in the cryostat chamber;
• The slides were then placed in a slidebox within the chamber until all sectioning
was complete;
• At the end of sectioning the slidebox was transferred to the -80 C freezer;
• For preparation the tissue sections were lyophilized in a freeze drier immediately
from the -80 C freezer.
To ensure reducibility and minimize the impact of any systematic source of bias the
experiments were performed in biological triplicate and the MSI data acquisition from
the 3xTg and WT mice performed in a pairwise manner (3xTg-WT etc); each slide
contained one 3xTg and one WT tissue section, thus were simultaneously prepared and
measured during the same MSI run. After MSI data acquisition the matrix was washed
off with 70 % ethanol and the tissue samples stained with cresyl violet (Nissl stain).
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Histological images were scanned with a MIRAX DESK digital slide scanner (3D
Histech, Hungary).
Protein MSI
The tissues were first washed in ice-cold isopropanol to fix the tissues and remove salts
and lipids. The washed tissue sections were then allowed to dry in air prior to matrix
deposition using the ImagePrep (Bruker Daltonics, Bremen, Germany) device. A
solution of 20 mg/mL SA in 70 % isopropanol with 0.1 % TFA was used as organic
matrix. MALDI MSI experiments were then performed using an Autoflex III MALDI-
linear-TOF (Bruker Daltonics), 100-200 µm pixel size, and 600 laser shots per pixel (50
laser shots per position of a random walk within each pixel). Positively charged ions
between m/z 2000 and 25000 were detected. Data acquisition, pre-processing (mass
spectral smoothing, baseline subtraction and total-ion-count normalization) and data
visualization were performed using the Flex software suite (FlexControl 3.0,
FlexAnalysis 3.0, FlexImaging 2.1).
Metabolite MSI
A uniform coating of 9-AA was added using the ImagePrep and a solution of 10 mg/mL
in 70 % MeOH. Metabolite MSI experiments were performed using a 9.4 T Apex Q
MALDI-FTICR (Bruker Daltonics) in negative ion mode, using a 50-150 µm pixel size,
in the range 50-1000 m/z by averaging signals from 600 laser shots per pixel (50 laser
shots per position of a random walk within each pixel; random walk enabled through
adapting the experiment pulse program). Each pixel’s mass spectrum was recalibrated
using the matrix peak of 9-AA (m/z 193,0771219) as an internal lock mass and
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normalized using the root-mean-square method. Data acquisition, pre-processing and
data visualization were performed using the Flex software suite (Compass 1.3,
FlexControl 3.0, FlexImaging 2.1).
MSI datasets contain an individual mass spectrum for each pixel. The ultrahigh mass
resolution of FTICR mass spectrometry generates large files and, thus, very large MSI
datasets (often >20 GB for a single tissue). An automated feature identification and
extraction routine was applied to extract the images of the detected peaks, thereby
reducing the data load by approximately a thousand fold [43].
MSI data filtering and pathway analysis
To further focus the analysis on known metabolites and to introduce metabolic pathway
information, the reduced MALDI MSI datasets were filtered using the Human
Metabolome Database. For each entry in the database, the compound names, exact
masses (converted to [M-H]- ions), and pathway memberships were extracted. Only
metabolite ions that corresponded to [M-H]- ions, within a mass tolerance of 1 ppm, and
only those whose isotopic profile matched that of the theoretical distribution (Pearson
correlation > 0.95, performed in Matlab) were retained.
Validation of proteins by immunohistochemistry
For all the procedure described below Phosphate Buffered saline (PBS) at 100 mM was
used. Frozen mouse brain sections were post-fixed in paraformaldehyde (4 % in PBS)
for 30 min and washed (6×5 min) in PBS. Sections were incubated for 30 min in 0.1 %
Triton X-100 containing 2 % normal donkey serum in PBS to block non-specific
binding. Without further washing, sections were incubated overnight at 4 °C with Anti-
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Neurogranin antibody (Abcam ab23570, 1:500) in 2 % normal donkey serum/0.1 %
Triton X-100 in PBS. Sections were then washed (3×10 min) in PBS and incubated for
2 h at room temperature with secondary antibody (AlexaFluor 594 nm donkey anti-
rabbit IgG, Molecular Probes, Invitrogen, 1:500) in 2 % normal donkey serum/0.1 %
Triton X-100 in PBS) and then washed again (3×10 min) in PBS. Sections were
mounted with a Prolong Antifade kit (Molecular Probes, Invitrogen) with DAPI.
Sections incubated without the primary antibodies served as negative controls.
Images were collected on a Olympus BX51 upright microscope using a 4×/0.13,
10×/0.30 or 40×/0.50 UPlanFLN objectives and captured using a Coolsnap ES camera
(Photometrics) through MetaVue Software (Molecular Devices). Images were then
processed and analysed using ImageJ (http://rsb.info.nih.gov/ij).
Results
The non-targeted nature of MSI led us to investigate whether it could be used to
investigate the chemical and spatial extent of the molecular disturbances occurring in an
Alzheimer’s disease rodent model. MSI is able to analyse different molecular classes by
changing sample preparation strategies and optimizing the mass spectrometer for the
mass range of the molecular class of interest. Furthermore, by combining MSI with
histology the differential MS profiles found in specific histopathological entities can be
used to identify candidate biomarkers [44].
Visualization of protein changes in Alzheimer’s disease mouse brain
MSI is a key technique in the visualization of intact proteins in tissue [45]. To detect
protein signals associated with Alzheimer’s disease, protein profiles from mouse brain
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sagittal tissue sections from three WT and three 3×Tg mice were acquired by MALDI-
MSI using SA as a matrix. Figure 1a) shows a typical average mass spectrum obtained
from a single WT mouse brain sagittal tissue section (green line), a single 3×Tg mouse
brain tissue section (red line), and the average mass spectrum of the entire data set
(purple line). A comparison of the WT and 3xTg average mass spectra revealed that
while many peaks were identical, two signals with m/z values of 7560 and 7870 showed
a much higher intensity in the spectra from WT mouse brains. Neurogranin is a small
78aa [46] post-synaptic protein. The peak at m/z 7560 was tentatively assigned to (the
sodium adduct of) neurogranin from the literature [46]. The distribution of this
neurogranin peak in the brain is shown in Figure 1b) in green. The peak at m/z 6400
corresponding to PEP-19 (in blue) and the peak at m/z 14200 corresponding to myelin
basic protein (in red) are shown as morphological references. Neurogranin is mainly
detected in healthy brain sections, being localized in the isocortex (indicated as area “a”
in the brain scheme), in particular in the prelimbic, somatomotor and anterior cingulate
areas. In order to ensure reproducibility, different animal pairs were analysed, showing
in all cases a m/z 7560 intensity difference between WT and 3×Tg mouse brain sections
(Figure 1.c). A t-test of the average intensity of the neurogranin MSI signal in the
isocortex, and using the Benjamini-Hochberg multiple testing correction, confirmed the
statistical significance of the detected changes, p<0.01. For a final validation, the
presence and distribution of the protein neurogranin was confirmed by
immunohistochemistry (IHC), as shown in Figure 1.d. No detectable signal was
observed for brain sections incubated without the primary antibody.
Visualization of metabolites changes in Alzheimer’s disease mouse brain
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For fundamental reasons, metabolomics is necessarily more sensitive than is proteomics
for detecting biochemical changes [47]. Metabolite MSI offers enormous clinical
potential by enabling molecule-specific imaging of a class of biomolecules for which
alternative histological stains typically lack molecular specificity, e.g. Oil Red stains
lipids and trialycerides. Furthermore, when combined with known metabolic pathways
[48], metabolite MSI provides a means to image the activities of pathways in tissues
directly. The matrix used for metabolite analysis was 9-AA, commonly used due to the
low intensity of matrix background ions [49]. The MS data acquisition was performed
on a 9.4T FTICR mass spectrometer, which provides routine ultrahigh mass resolution
and accurate mass in the low m/z range, enabling each mass spectral peak to be fully
resolved. The assignments were based on accurate mass. By using known pathway
information, it was observed that control and Alzheimer’s disease mouse brain sagittal
sections showed highly distinct metabolic signatures. Figure 2 shows the purine
metabolic pathway, indicating the distributions of detected ions within this pathway for
WT and 3×Tg mice. By using MALDI-MSI we were able to detect a number of the
constituent metabolites, although others were below the limit of detection. Figure 2
shows several clear differences in metabolite concentration between WT and 3×Tg
mouse brain sections. The concentration of adenine and AMP are higher in the WT
mouse, while other metabolites like inosine, hypoxanthine, xanthine, and uric acid are
increased in the 3×Tg mouse. Furthermore, ascorbic acid, albeit not in the purine
metabolic ‘pathway’, shows a much higher abundance in WT brain sections.
Apart from the purine metabolic pathway, and as shown for ascorbic acid, the
concentrations of some other metabolites appear to be altered in the case of 3×Tg mouse
brain. As shown in Figure 3, when mass spectra of both healthy and AD mouse brains
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were compared, a peak at m/z 410.2373 appeared to be a lot more intense for AD brain.
For the identity assignment of this peak, the METLIN database was chosen. This a
freely accessible web-based data repository, that has been developed to assist in
metabolite research and to facilitate metabolite identification through mass analysis
[50]. The mass of the peak was assigned, with a mass tolerance of < 1 ppm, to N-
arachidonoyl taurine, a fatty acyl amide of the amino acid taurine. The tissue
distribution of N-arachidonyl taurine in brain sections imaged at 150 µm pixel center-
to-pixel center distance is also shown in Figure 3 (in green). The peak at m/z 885.6,
corresponding to phosphatidyl inositol (18:0) (in red), is shown as a morphological
reference. As shown in the 50 µm resolution image, N-arachidonyl taurine (in orange) is
mainly detected in the 3×Tg brain, and localized in the hippocampal region. However, it
is not homogeneously distributed, showing higher intensities in the stratum oriens and
dentate gyrus regions, and lower intensities in the pyramidal layer and stratum radiatum
regions.
Discussion
MALDI MSI has several distinct advantages for neuroscience and neuropathological
research. MALDI MSI provides simultaneous label free, multiplex imaging of
endogenous biomolecules. Using essentially the same technology but different sample
preparation methods MALDI MSI may be used to analyze peptides, proteins, lipids,
metabolites, neurotransmitters and even N-linked glycans; for several of which there
exists no generally applicable method, analogous to immunohistochemistry, that may be
used to record molecule-specific distributions. Furthermore, any enzymatic/metabolic
change that results in a change in mass can be resolved. For example MALDI MSI has
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detected changes in post-translational modifications in neuropeptides and proteins and
even, via the introduction of 13C-labeled glucose, traced metabolic flux (Sugiura et al.,
2014).
MALDI MSI data is routinely registered to histology, and has been registered to both
fluorescence microscopy and magnetic resonance imaging, thus enabling cell specific
molecular profiles to be obtained, within their correct histopathological context, and to
investigate/utilize associations with in-vivo imaging methods. These capabilities are
particularly suited to discovery research, such as reported here, which benefit from the
broad molecular scope of the analysis for the discovery of previously unknown
molecular changes.
The literature on many diseases is data-rich and tested-hypothesis-poor. Given the
essential lack of treatments available, the dementias largely fall into this class. The
issues are compounded by (i) the difficulty of assessing cognitive function accurately,
and (ii) that (in the case of genuine Alzheimer’s in humans) a definitive diagnosis is
possible solely post mortem. Thus data-driven strategies that seek to discriminate
patients from controls are appropriate, as they can give strong indications as to which
surrogate variables are different in disease vs control samples. One hypothesis is then
that restoring the variable to their ‘normal’ range might be of therapeutic benefit, but
discovering the chief differences is necessarily the first need. Mass spectrometry
imaging strategies represent an exceptionally useful strategy for discovering such
markers, and were applied herein to assess differences between brain slices taken from
3×Tg mice and wild-type controls. We analysed both the proteome and the metabolome.
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In the present case, we discovered a major change in the AD proteome, in that a peak at
m/z 7560 assigned to the post-synaptic protein neurogranin (Ng), and confirmed by
immunohistochemistry, was significantly down-regulated in the 3×Tg mouse. This
finding is in agreement with previous reports, which have shown a loss of Ng with
normal ageing in mice [51], in the AD mouse model Tg2576 [52] and in the brains of
AD patients by western blot and immunohistochemistry [53, 54]. Conversely Ng has
been found to be increased in the CSF of Alzheimer’s patients [55-60]. The levels of
proteins in brain parenchyma can correlate positively or negatively with levels of
proteins in CSF: increased production of pTau in AD brain results in increased levels in
CSF, while increased aggregation of A40-42 in AD brains results in decreased levels of
A in CSF; we can therefore hypothesise that neuronal loss results in decreased Ng
immunohistochemical staining and release of neurogranin in to the interstitial fluid and
CSF [61].
Even more interesting and novel were the findings that purine metabolism was seriously
deregulated, with substantial decreases in the concentration of adenine and AMP but
with other metabolites like inosine, hypoxanthine, xanthine, and uric acid being
noticeably increased in the 3×Tg mouse. Furthermore, ascorbic acid was much
decreased in the 3×Tg mouse. These findings indicate that the care taken to limit post-
mortem degradation of metabolites during the excision of the mouse brains, as well as
their sectioning, enabled the detection of hydrophilic small molecules that differ
reproducibly and discernibly between samples. Furthermore, these differences are
consistent with known AD pathology.
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There is considerable evidence for the roles of purinergic signalling [62], and especially
the role of uric acid in Alzheimer’s disease (Kaddurah-Daouk et al., 2013; McFarland et
al., 2013) and in a variety of kinds of inflammatory process, including peanut allergy
[63, 64], pro-inflammatory cytokine production [65, 66], the Plasmodium falciparum-
induced inflammatory response [67] and the mechanistic basis for the action of alum as
an adjuvant [68]. Adenosine and inosine mediate anti-inflammatory effects via A2 and
A3 receptors [69, 70] (and IL-1β feeds back thereupon [71]), while purine metabolism
contributes to the anti-inflammatory action of aspirin [72]. The general consonance
between the metabolic ‘signature’ (i.e. those metabolites noticeably up- and down-
regulated in both the 3×Tg mouse and the hyperallergic response [64] is especially
striking, implying a similar overall regulation. We have also shown experimentally that
uric acid is raised significantly following heart failure [73]
Although a raised uric acid in serum is associated with a lower likelihood of AD and
dementia [74, 75], its relation to brain levels of uric acid is unknown.
The loss of ascorbate in the 3×Tg mouse is consistent with the well-known oxidative
stress accompanying, and probably contributing to, AD [76]. What has not been seen
previously is the derangement of purine metabolism that we report here. It would seem
that purine metabolism forms an important link with cytokine signalling as part of
neuroinflammation. Given the role of neuroinflammation [77, 78] and diet [79] in
exacerbating AD and other neurodegenerative disorders, this warrants considerable
further investigation.
Acknowledgments
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DBK thanks the Biotechnology and Biological Sciences Research Council (grant
BB/L025752/1) for support. This is also a contribution from the Manchester Centre for
Synthetic Biology of Fine and Speciality Chemicals (SYNBIOCHEM) (BBSRC grant
BB/M017702/1). CE and LAM thank the support of the ZonMW Zenith project
“Imaging Mass Spectrometry-Based Molecular Histology: Differentiation and
Characterization of Clinically Challenging Soft Tissue Sarcomas” (No. 93512002;
B.H.) and the ICT consortium COMMIT project “e-biobanking with Imaging”.
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Figure captions
Figure 1. a) Average MALDI-TOF-MSI spectra from a single WT mouse (green), a
single 3×Tg mouse (red) and the average spectrum of all animals (purple). Differentially
expressed m/z species are marked by arrows. b) MALDI-TOF-MSI overview image
visualizing 7.5 kDa Neurogranin (green), PEP-19 (blue) and 14.2 kDa myelin basic
protein (red); scale bar = 2mm. A scheme of a sagittal brain section is also included to
highlight the a: isocortex, b: fibre tracts, c: thalamus, d: hippocampus, and e: midbrain,
pons, medulla and fibre tracts. c) Visualization of m/z 7560 in a MALDI-TOF-MSI
reproducibility study based on the analysis of four animal pairs. d)
Immunohistochemical validation of Neurogranin, scale bar 400 μm.
Figure 2. MALDI-FTICR-MSI visualization of accurate mass (<1 ppm) and isotope
profile filtered (Pearson correlation > 0.95) metabolites involved the purine metabolic
pathway for WT and 3×Tg mice. All tissue sections are sagittal with the cerebellum
located at the top. Scale bar = 2 mm.
Figure 3. MALDI-FTICR-MS spectra from WT mouse (blue) and 3×Tg mouse (red) for
the m/z range 405-412. MSI visualization of N-arachidonoyl taurine (green) in WT and
3×Tg sagittal mouse brain tissue sections at 150 µm resolution. Phosphatidylinositol
(18:0, 20:2) (red) is also shown to outline the structure and orientation of the sections.
The bottom right MSI image shows a higher mass resolution, 50 µm pixel size, MALDI
MSI analysis of the area indicated by the white square.
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Graphic abstract
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Highlights
Protein and metabolite MALDI MSI comparison of AD transgenic mice with
wild type.
Independently validated differences in protein expression in AD transgenic
mice.
Metabolic differences in AD transgenic mice consistent with known AD
biology.
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