Correlative chemical imaging identifies amyloid peptide signatures of neuritic plaques and dystrophy in human sporadic Alzheimer's disease

OBJECTIVE: Alzheimer's disease (AD) is the most common neurodegenerative disease. The predominantly sporadic form of AD (sAD) is age-related, but the underlying pathogenic mechanisms remain not fully understood. Current efforts to combat the disease focus on the main pathological hallmarks, in particular beta-amyloid (Aβ) plaque pathology. According to the amyloid cascade hypothesis, Aβ is the critical early initiator of AD pathogenesis. Plaque pathology is very heterogeneous, where a subset of plaques, neuritic plaques, are considered most neurotoxic rendering their in depth characterization essential to understand Aβ pathogenicity. METHODS: To delineate the chemical traits specific to neuritic plaque types, we investigated senile Aβ pathology in post mortem human sporadic AD brain using advanced correlative biochemical imaging based on immunofluorescence microscopy and mass spectrometry imaging (MSI). RESULTS: Immunostaining-guided MSI identified distinct Aβ signatures of neuritic plaques characterized by increased Aβ 1-42(ox) and Aβ2-42. Moreover, correlation with a marker of dystrophy (reticulon 3, RTN3) identified key Aβ species that both delineate neuritic plaques and display association with neuritic dystrophy. CONCLUSION: Together these correlative imaging data shed light on the complex biochemical architecture of neuritic plaques and associated dystrophic neurites. These in turn are obvious targets for disease-modifying treatment strategies, as well as novel biomarkers of Aβ pathogenicity

Chemical traits of cerebral amyloid angiopathy in familial British-, Danish-, and non-Alzheimerʼs dementias

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant forms of dementia caused by mutations in the integral membrane protein 2B (ITM2B, also known as BRI2) gene. Secretase processing of mutant BRI2 leads to secretion and deposition of BRI2-derived amyloidogenic peptides, ABri and ADan that resemble APP/β-amyloid (Aβ) pathology, which is characteristic of Alzheimer's disease (AD). Amyloid pathology in FBD/FDD manifests itself predominantly in the microvasculature by ABri/ADan containing cerebral amyloid angiopathy (CAA). While ABri and ADan peptide sequences differ only in a few C-terminal amino acids, CAA in FDD is characterized by co-aggregation of ADan with Aβ, while in contrast no Aβ deposition is observed in FBD. The fact that FDD patients display an earlier and more severe disease onset than FBD suggests a potential role of ADan and Aβ co-aggregation that promotes a more rapid disease progression in FDD compared to FBD. It is therefore critical to delineate the chemical signatures of amyloid aggregation in these two vascular dementias. This in turn will increase the knowledge on the pathophysiology of these diseases and the pathogenic role of heterogenous amyloid peptide interactions and deposition, respectively. Herein, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in combination with hyperspectral, confocal microscopy based on luminescent conjugated oligothiophene probes (LCO) to delineate the structural traits and associated amyloid peptide patterns of single CAA in postmortem brain tissue of patients with FBD, FDD as well as sporadic CAA without AD (CAA+) that show pronounced CAA without parenchymal plaques. The results show that CAA in both FBD and FDD consist of N-terminally truncated- and pyroglutamate-modified amyloid peptide species (ADan and ABri), but that ADan peptides in FDD are also extensively C-terminally truncated as compared to ABri in FBD, which contributes to hydrophobicity of ADan species. Further, CAA in FDD showed co-deposition with Aβ x-42 and Aβ x-40 species. CAA+ vessels were structurally more mature than FDD/FBD CAA and contained significant amounts of pyroglutamated Aβ. When compared with FDD, Aβ in CAA+ showed more C-terminal and less N-terminally truncations. In FDD, ADan showed spatial co-localization with Aβ3pE-40 and Aβ3-40 but not with Aβx-42 species. This suggests an increased aggregation propensity of Aβ in FDD that promotes co-aggregation of both Aβ and ADan. Further, CAA maturity appears to be mainly governed by Aβ content based on the significantly higher 500/580 patterns observed in CAA+ than in FDD and FBD, respectively. Together this is the first study of its kind on comprehensive delineation of Bri2 and APP-derived amyloid peptides in single vascular plaques in both FDD/FBD and sporadic CAA that provides new insight in non-AD-related vascular amyloid pathology. (Figure presented.

Correlative Chemical Imaging and Spatial Chemometrics Delineate Alzheimer Plaque Heterogeneity at High Resolution

We present a novel, correlative chemical imaging strategy based on multimodal matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), hyperspectral microscopy, and spatial chemometrics. Our workflow overcomes challenges associated with correlative MSI data acquisition and alignment by implementing 1+1-evolutionary image registration for precise geometric alignment of multimodal imaging data. This enabled multivariate statistical modeling of multimodal imaging data using a novel multiblock orthogonal component analysis approach to identify covariations of biochemical signatures between and within imaging modalities at MSI pixel resolution. We demonstrate the method’s potential through its application towards delineating chemical traits of Alzheimer’s disease (AD) pathology. Here, trimodal MALDI MSI of transgenic AD mouse brain delineates beta-amyloid (Aβ) plaque-associated co-localization of lipids and Aβ peptides. Finally, we establish an improved image fusion approach for correlative MSI and functional amyloid microscopy. This allowed high resolution prediction of correlative, multimodal MSI signatures towards distinct amyloid structures within single plaque features critically implicated in Aβ pathogenicity

Chemical imaging of sphingolipids and phospholipids at the single amyloid-β plaque level in post-mortem human Alzheimer’s disease brain

Lipids dysregulations have been critically implicated in Alzheimer’s disease (AD) pathology. Chemical analysis of amyloid-β (Aβ) plaque pathology in transgenic AD mouse models has demonstrated alterations in the microenvironment in direct proximity to Aβ plaque pathology. In mouse studies, differences in lipid patterns linked to structural polymorphism among Aβ pathology, such as diffuse, immature, and mature fibrillary aggregate have also been reported. To date, no comprehensive analysis of neuronal lipids microenvironment changes in human AD tissue has been performed. Here, for the first time we leverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) though high speed and spatial resolution commercial time-of-light instrument, as well as high mass resolution in-house developed orbitrap system to characterize the lipid microenvironment in postmortem human brain tissue from AD patients carrying Presenilin 1 mutations (PSEN 1) that lead to familial forms of AD (fAD). Interrogation of the spatially resolved MSI data on a single Aβ plaque allowed us to verify nearly 40 sphingolipid and phospholipid species from diverse subclasses being enriched and depleted in relation to the Aβ deposits. This included monosialo-gangliosides (GM), ceramide monohexosides (HexCer), ceramide-1-phosphates (CerP), ceramide phosphoethanolamine conjugates (PE-Cer), sulfatides (ST), as well as phosphatidylinositols (PI), phosphatidylethanolamines (PE), and phosphatidic acid (PA) species (including Lyso-forms). Indeed, many of the sphingolipids species overlap with the species previously seen in transgenic AD mouse models. Interestingly, in comparison to the animal studies, we observed an increased localization of PE and PI species containing arachidonic acid (AA). These finding are highly relevant, demonstrating for the first time Aβ plaque pathology-related alteration in the lipid microenvironment in humans. They provide a basis for development of potential lipid biomarkers for AD characterization and insight into human-specific molecular pathway alterations

Amyloid Plaque Polymorphism is Associated with Distinct Lipid Accumulations Revealed by Trapped Ion Mobility Mass Spectrometry Imaging (TIMS MSI)

Understanding of Alzheimer’s disease (AD) pathophysiology, requires molecular assessment of how key pathological factors, specifically amyloid β (Aβ) plaques, influence the surrounding microenvironment. Here, neuronal lipids are particularly of interest as these are implicated in pathological- and neurodegenerative processes in AD. The exact molecular characteristics of the cellular environment in direct proximity to Aβ plaques are however still not known, not in the least due to high molecular complexity of lipid species but also due to the lacking spatial resolution, sensitivity, and specificity of analytical approaches. Likewise, how such micro environmental changes differ, across structurally polymorphic Aβ features - such as diffuse, immature and mature, fibrillary structures - has been a challenge, requiring complemental, multimodal imaging approaches. Herein, we used matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) trapped ion mobility spectrometry Time-of-Flight (TIMS TOF) in combination with hyperspectral microscopy to probe lipidomic microenvironment associated with structural polymorphism of Aβ plaque in transgenic mouse model of Alzheimer’s disease (tgAPPSWE).Integrated multivariate imaging data analysis revealed alteration of multiple lipid species showing a general, Aβ associated enrichment/depletion patterns. The hyperspectral imaging strategy further delineated unique distribution of PA, PE-Cer and PI lipids to more/less aggregated Aβ fibrillary structures present within individual Aβ plaques at different timepoints of progressing plaque pathology. Using an elaborate on tissue and ex situ validation approach, the unique possibility to obtain gas-phase isobar and isomer separations through TIMS TOF, facilitated unambiguous identification of lipid isomers that showed plaque pathology associated localizations. Finally, we followed AD pathology associated lipid changes over time, identifying plaque growth and maturation to be characterized by peripheral accumulation of PI (40:6). Together, these data demonstrate the potential of multimodal imaging approaches to overcome limitations associated with conventional advanced MS imaging applications. This allowed for differentiation of both distinct lipid components in a complex micro environment, as well as their correlation to disease relevant amyloid plaque polymorphs