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Delineating Amyloid Plaque Associated Neuronal Sphingolipids In Transgenic Alzheimer’s Disease Mice (tgArcSwe) Using MALDI Imaging Mass Spectrometry Ibrahim Kaya, Dimitri Brinet, Wojciech Michno, Stina Syvanen, Dag Sehlin, Henrik Zetterberg, Kaj Blennow, and Jörg Hanrieder ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00391 • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 18, 2016
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ACS Chemical Neuroscience
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Delineating Amyloid Plaque Associated Neuronal Sphingolipids in
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transgenic Alzheimer’s Disease Mice (tgArcSwe) Using MALDI
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Imaging Mass Spectrometry
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Ibrahim Kaya1#, Dimitri Brinet1,2#, Wojciech Michno1, Stina Syvänen3, Dag Sehlin3,
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Henrik Zetterberg1,4,5, Kaj Blennow1,4, and Jörg Hanrieder1,5,6
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1
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Gothenburg, Mölndal, Sweden
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2
Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden
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3
Department of Public Health and Caring Sciences, Uppsala University, Uppsala, Sweden
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Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden
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5
Department of Molecular Neuroscience, UCL Institute of Neurology, University College
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London, UK
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6
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Gothenburg, Sweden
Department of Psychiatry and Neurochemistry, Sahlgrenska Academy at the University of
Department of Chemistry and Chemical Engineering, Chalmers University of Technology,
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16 17
Corresponding Author:
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Jörg Hanrieder, PhD
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Department of Psychiatry and Neurochemistry, Sahlgrenska Academy at the University of
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Gothenburg, Mölndal Hospital, House V, Biskopsbogatan 27, SE-43180 Mölndal, Sweden
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[email protected]; +46-31-343 2377
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ABSTRACT
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The major pathological hallmarks of Alzheimer’s disease (AD) are the progressive
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aggregation and accumulation of beta-amyloid (Aβ) and hyperphosphorylated tau protein into
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neurotoxic deposits. Aβ aggregation has been suggested as the critical early inducer, driving
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the disease progression. However, the factors that promote neurotoxic Aβ aggregation
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remain
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comprehensively elucidate the spatial distribution patterns of lipids, peptides and proteins in
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biological tissue sections. In the present study, matrix-assisted laser desorption/ionization
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(MALDI) mass spectrometry (MS)-based imaging was used on transgenic Alzheimer’s
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disease mouse (tgArcSwe) brain tissue to investigate the sphingolipid microenvironment of
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individual Aβ plaques and elucidate plaque-associated sphingolipid alterations. Multivariate
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data analysis was used to interrogate the IMS data for identifying pathologically relevant,
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anatomical features based on their lipid chemical profile. This approach revealed sphingolipid
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species that distinctly located to cortical and hippocampal deposits, whose Aβ identity was
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further verified using fluorescent amyloid staining and immunohistochemistry. Subsequent
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multivariate statistical analysis of the spectral data revealed significant localization of
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gangliosides and ceramides species to Aβ positive plaques, which was accompanied by
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distinct local reduction of sulfatides. These plaque-associated changes in sphingolipids levels
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implicate a functional role of sphingolipid metabolism in Aβ plaque pathology and AD
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pathogenesis. Taken together, the presented data highlight the potential of imaging mass
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spectrometry as a powerful approach for probing Aβ plaque-associated lipid changes
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induced by AD neuropathology.
elusive.
Imaging
mass
spectrometry
(IMS)
is
a
powerful
technique
to
46
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Keywords: Alzheimer’s disease, Amyloid-β plaque pathology, MALDI Imaging mass
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spectrometry, sphingolipids, tgArcSwe.
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INTRODUCTION
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Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. The
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neuropathology of AD is characterized by the formation of protein deposits in the brain
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including intercellular neurofibrillary tangles consisting of hyper-phosphorylated tau protein1
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and extracellular plaques formed by aggregated amyloid-β (Aβ) peptides
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rapidly aggregate to oligomers, protofibrils and fibrils, eventually leading to formation of
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extracellular plaques. Little is known about the molecular mechanisms of how monomeric Aβ
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peptides are converted to neurotoxic, aggregated forms that are rich in β-sheet motifs. A
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number of biochemical and clinical studies suggest that in addition to peptide centric
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mechanisms, changes in neuronal lipid metabolism may be implicated in AD pathogenesis
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and plaque pathology in particular.4,
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apolipoprotein E (APOE) ε4 allele, a lipid transporter protein, was identified as the major risk
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factor to develop sporadic AD, further suggesting a possible role for lipids in AD
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pathogenesis6-9. Hence, several studies on the relevance of neuronal lipid species
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associated with AD pathology, including sphingolipids10, 11, cholesterol12 and phospholipids13
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have been reported. In particular, a potential role in AD pathogenesis has been suggested
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for a number of sphingolipid species, including ceramides7,
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gangliosides16-19. Therefore, in order to further delineate the relevance of sphingolipids in AD
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pathogenesis, it is important to outline their spatial distribution profile in situ. Genetically
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altered mouse models are of central relevance to probe the molecular mechanisms of AD
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pathology.20, 21 For instance, a transgenic mouse model of AD, with mice carrying both the
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Arctic (E693G) and the Swedish (K670M/N671L) mutations (tgArcSwe) of amyloid precursor
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protein (APP) has been developed. These mice exhibit in extensive Aβ deposition with an
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onset at the age of 5–6 months, and are a well-suited model system to study molecular
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mechanisms for Aβ deposition.22
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In order to characterize disease pathology in situ, advanced molecular imaging techniques
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are required including imaging mass spectrometry (IMS). This technique is often referred as
5
2, 3
. Aβ peptides
Furthermore, genetic predisposition with the
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, sulfatides14,
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and
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molecular histology and allows to generate spatial intensity distribution maps of molecular
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species
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desorption/ionization (MALDI) based IMS is a well suited IMS modality to probe neuronal
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lipids24, including gangliosides25 as well as for in situ characterization of endogenous
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neuropeptides26,
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IMS, has been previously successfully demonstrated for probing Aβ peptide pathology in
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tgArcSwe mice.28
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In
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microenvironment, including ganglioside-, sulfatide- and ceramide- localizations to cortical
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and hippocampal Aβ plaques in tgArcSwe mice. Here, IMS was used in conjunction with
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multivariate statistical analysis (MVSA) tools to identify regional plaque-associated changes
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in neuronal sphingolipid chemistry.
the
in
complex
27
present
biological
tissues
23
.
In
particular,
matrix
assisted
laser
associated with neurodegenerative disease pathology. Indeed, MALDI-
study,
MALDI-IMS
was
employed
to
examine
the
sphingolipid
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RESULTS AND DISCUSSION
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MALDI Imaging MS Reveals Distinct Localization of Neuronal Sphingolipid Species on
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the Cortical and Hippocampal Amyloid-β Plaques
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In MALDI-IMS, sample preparation includes deposition of a matrix species, dissolved in an
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aqueous/organic solvent system onto the tissue sections. However, this process can result in
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lateral analyte diffusion and disturbance of tissue morphology. In contrast, sublimation is a
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solvent free approach for matrix deposition that overcomes analyte delocalization issues
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arising with wet matrix application and hence allows for high-spatial resolution
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sublimation of 1,5-diaminonaphthalene (1,5-DAN) for MALDI IMS was shown to give
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enhanced signal intensity for neuronal lipid species at high spatial resolution, particularly in 30
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. Previously,
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negative ion mode
. In the present study, 1,5-DAN sublimation was therefore used for
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MALDI-IMS analysis of neuronal sphingolipids in tgArcSwe mice. The acquired image data
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were investigated by using unsupervised multivariate statistics based on hierarchical
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clustering analysis (bisecting k-means) in order to obtain image segmentation of anatomical
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regions of interest based on their lipid chemical profile. Here, image segmentation identified
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deposit-like features in the cortex and hippocampus (Figure 1A,B). These features were
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assigned as individual regions of interest (ROI) and correlated to the processed MS data, in
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order to identify the associated chemical species that allowed image segmentation of the
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regions.
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Figure 1. Multivariate image analysis of MALDI-IMS data reveals cortical and hippocampal sphingolipid accumulations in tgArcSwe mouse brain. (A) Bright field image showing dorsolateral part of a coronal section used for IMS analysis. Anatomical regions wre annotated including: Ctx cortex, WM white matter, LV lateral ventricle, hipp hippocampus, DG dentate gyrus of the hippocampus. (B) Segmentation map obtained from multivariate image analysis of the MALDI-IMS data. Bisecting k-means based hierarchical clustering analysis identified plaque-like features (green, yellow, orange) in the cortex and hippocampus based on the encoded chemical information. Inspection of the associated variables (m/z values) expressing the clustering behavior, revealed m/z values localizing to anatomical features. (C) This includes e.g. Cer(d18:1/12:0) at m/z 480.5 as further verified by the corresponding single ion image as a representative plaque associate sphingolipid species. Scale bar = 0.5 mm.
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species to the plaque-like features, including ceramides and gangliosides. Visualization of
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single ion intensity distributions (i.e. single ion images) for the individual ceramide- (e.g.
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Cer(d18:1/12:0), m/z 480.5, Figure 1C) and ganglioside species (Figure 4) showed a
Inspection of the corresponding variables revealed localization of various sphingolipid
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consistent deposit-like distribution patterns throughout the cortical and hippocampal regions.
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Hence, the here employed workflow for unbiased segmentation of the complex imaging data
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was found to be a strong approach for elucidating the chemical composition of the plaque-
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like deposits.
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Amyloid-β
Plaque
Identification
and
Validation
by
Subsequent
Fluorescent
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Histochemical Amyloid Staining
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In order to verify the potential plaque identity as observed for individual ceramide- and
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ganglioside accumulations, including e.g. Cer(d18:1/12:0) (Figure 2A, B), histochemical
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staining was performed using a luminescent conjugated oligothiophene (LCO), h-FTAA, a
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fluorescent amyloid probe (Figure 2C)
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amyloid staining and distributions profiles of distinct sphingolipid species as identified by
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MALDI-IMS (Figure 2D). Furthermore, in order to confirm the specificity of the chemical
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amyloid staining to Aβ fibrils, double staining of h-FTAA and Aβ immunohistochemistry (IHC)
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was performed using a monoclonal Aβ antibody (Aβ1-16, 6E10). The fluorescent imaging
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results show clear double positive staining of the plaques for both h-FTAA and Aβ, thereby
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confirming the Aβ identity of the h-FTAA stained deposits (Suppl. Figure S-1A-C).
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. Here, a strong co-localization was observed for
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Figure 2. Identification of Aβ deposits localized with MALDI-IMS using fluorescent
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amyloid staining. (A) Single ion image of m/z 480.5 (Cer(d18:1/12:0)) using MALDI-IMS
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with 10 µm spatial resolution. (B) Magnified single ion images of m/z 480.5 from the cortex.
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(C) Histologically identified plaque, stained with h-FTAA reveals Aβ fibril structures in the
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cortical regions (D) co-localized with the accumulation observed in the magnified single ion
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images of m/z 480.5. Scale bars A=400 µm, B-D= 100 µm.
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In addition, to further verify the lipid localizations to amyloid plaques, a bottom up
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multivariate, correlation analysis approach was used. Here, both imaging MS data and
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fluorescent staining data (h-FTAA and IHC) were co-registered in the SciLs software (Suppl.
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Figure S-1D,E). This was followed by annotation of h-FTAA/Aβ double positive features in
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the fluorescence imaging data and correlation analysis to the whole imaging data set. The
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results revealed the most prominent variables (m/z values) that correlate with Aβ and h-
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FTAA double positive plaque regions (Suppl. Figure S-1D,E). Here, the most significant
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peaks above a certain threshold at p
