Correlative Chemical Imaging and Spatial Chemometrics Delineate Alzheimer Plaque Heterogeneity at High Spatial 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 and their integration in a common, truly multimodal imaging data matrix with maintained MSI resolution (10 µm). 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 toward delineating chemical traits of Alzheimer's disease (AD) pathology. Here, trimodal MALDI MSI of transgenic AD mouse brain delineates beta-amyloid (Aß) plaque-associated co-localization of lipids and Aß peptides. Finally, we establish an improved image fusion approach for correlative MSI and functional fluorescence microscopy. This allowed for high spatial resolution (300 nm) prediction of correlative, multimodal MSI signatures toward distinct amyloid structures within single plaque features critically implicated in Aß pathogenicity.

Structural amyloid plaque polymorphism is associated with distinct lipid accumulations revealed by trapped ion mobility mass spectrometry imaging.

Understanding of Alzheimer's disease (AD) pathophysiology requires molecular assessment of how key pathological factors, specifically amyloid ß (Aß) plaques, influence the surrounding microenvironment. Here, neuronal lipids have been implicated in Aß plaque pathology, though the lipid microenvironment in direct proximity to Aß plaques is still not fully resolved. A further challenge is the microenvironmental molecular heterogeneity, across structurally polymorphic Aß features, such as diffuse, immature, and mature, fibrillary aggregates, whose resolution requires the integration of advanced, multimodal chemical imaging tools. Herein, we used matrix-assisted laser desorption/ionization trapped ion mobility spectrometry time-of-flight based mass spectrometry imaging (MALDI TIMS TOF MSI) in combination with hyperspectral confocal microscopy to probe the lipidomic microenvironment associated with structural polymorphism of Aß plaques in transgenic Alzheimer's disease mice (tgAPPSWE ). Using on tissue and ex situ validation, TIMS MS/MS facilitated unambiguous identification of isobaric lipid species that showed plaque pathology-associated localizations. Integrated multivariate imaging data analysis revealed multiple, Aß plaque-enriched lipid patterns for gangliosides (GM), phosphoinositols (PI), phosphoethanolamines (PE), and phosphatidic acids (PA). Conversely, sulfatides (ST), cardiolipins (CL), and polyunsaturated fatty acid (PUFA)-conjugated phosphoserines (PS), and PE were depleted at plaques. Hyperspectral amyloid imaging further delineated the unique distribution of PA and PE species to mature plaque core regions, while PI, LPI, GM2 and GM3 lipids localized to immature Aß aggregates present within the periphery of Aß plaques. Finally, we followed AD pathology-associated lipid changes over time, identifying plaque- growth and maturation to be characterized by peripheral accumulation of PI (18:0/22:6). Together, these data demonstrate the potential of multimodal imaging approaches to overcome limitations associated with conventional advanced MS imaging applications. This allowed for the differentiation of both distinct lipid components in a complex micro-environment as well as their correlation to disease-relevant amyloid plaque polymorphs. Cover Image for this issue: https://doi.org/10.1111/jnc.15390.

Chemical imaging of evolving amyloid plaque pathology and associated Aß peptide aggregation in a transgenic mouse model of Alzheimer's disease.

One of the major hallmarks of Alzheimer's disease (AD) pathology is the formation of extracellular amyloid ß (Aß) plaques. While Aß has been suggested to be critical in inducing and, potentially, driving the disease, the molecular basis of AD pathogenesis is still under debate. Extracellular Aß plaque pathology manifests itself upon aggregation of distinct Aß peptides, resulting in morphologically different plaque morphotypes, including mainly diffuse and cored senile plaques. As plaque pathology precipitates long before any clinical symptoms occur, targeting the Aß aggregation processes provides a promising target for early interventions. However, the chain of events of when, where and what Aß species aggregate and form plaques remains unclear. The aim of this study was to investigate the potential of matrix-assisted laser desorption/ionization imaging mass spectrometry as a tool to study the evolving pathology in transgenic mouse models for AD. To that end, we used an emerging, chemical imaging modality - matrix-assisted laser desorption/ionization imaging mass spectrometry - that allows for delineating Aß aggregation with specificity at the single plaque level. We identified that plaque formation occurs first in cortical regions and that these younger plaques contain higher levels of 42 amino acid-long Aß (Aß1-42). Plaque maturation was found to be characterized by a relative increase in deposition of Aß1-40, which was associated with the appearance of a cored morphology for those plaques. Finally, other C-terminally truncated Aß species (Aß1-38 and Aß1-39) exhibited a similar aggregation pattern as Aß1-40, suggesting that these species have similar aggregation characteristics. These results suggest that initial plaque formation is seeded by Aß1-42; a process that is followed by plaque maturation upon deposition of Aß1-40 as well as deposition of other C-terminally modified Aß species.

Chemometric Strategies for Sensitive Annotation and Validation of Anatomical Regions of Interest in Complex Imaging Mass Spectrometry Data.

Imaging mass spectrometry (IMS) is a promising new chemical imaging modality that generates a large body of complex imaging data, which in turn can be approached using multivariate analysis approaches for image analysis and segmentation. Processing IMS raw data is critically important for proper data interpretation and has significant effects on the outcome of data analysis, in particular statistical modeling. Commonly, data processing methods are chosen based on rational motivations rather than comparative metrics, though no quantitative measures to assess and compare processing options have been suggested. We here present a data processing and analysis pipeline for IMS data interrogation, processing and ROI annotation, segmentation, and validation. This workflow includes (1) objective evaluation of processing methods for IMS datasets based on multivariate analysis using PCA. This was then followed by (2) ROI annotation and classification through region-based active contours (AC) segmentation based on the PCA component scores matrix. This provided class information for subsequent (3) OPLS-DA modeling to evaluate IMS data processing based on the quality metrics of their respective multivariate models and for robust quantification of ROI-specific signal localization. This workflow provides an unbiased strategy for sensitive annotation of anatomical regions of interest combined with quantitative comparison of processing procedures for multivariate analysis allowing robust ROI annotation and quantification of the associated molecular histology.

Discovery and development of substituted thiadiazoles as inhibitors of Staphylococcus aureus Sortase A.

High-throughput screening of small-molecule libraries has led to the identification of thiadiazoles as a new class of inhibitors against Staphylococcus aureus sortase A (SrtA). N-(5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide (IC50 = 3.8 µM) was identified as a potent inhibitor of SrtA after synthetic modification of hit compounds. Additional ligands developed in this study displayed affinities in the low micromolar range without affecting bacterial growth in vitro. The study also suggest a new mode of action through covalent binding to the active site cysteine.

Pyroglutamation of amyloid-ßx-42 (Aßx-42) followed by Aß1-40 deposition underlies plaque polymorphism in progressing Alzheimer's disease pathology.

Amyloid-ß (Aß) pathology in Alzheimer's disease (AD) is characterized by the formation of polymorphic deposits comprising diffuse and cored plaques. Because diffuse plaques are predominantly observed in cognitively unaffected, amyloid-positive (CU-AP) individuals, pathogenic conversion into cored plaques appears to be critical to AD pathogenesis. Herein, we identified the distinct Aß species associated with amyloid polymorphism in brain tissue from individuals with sporadic AD (s-AD) and CU-AP. To this end, we interrogated Aß polymorphism with amyloid conformation-sensitive dyes and a novel in situ MS paradigm for chemical characterization of hyperspectrally delineated plaque morphotypes. We found that maturation of diffuse into cored plaques correlated with increased Aß1-40 deposition. Using spatial in situ delineation with imaging MS (IMS), we show that Aß1-40 aggregates at the core structure of mature plaques, whereas Aß1-42 localizes to diffuse amyloid aggregates. Moreover, we observed that diffuse plaques have increased pyroglutamated Aßx-42 levels in s-AD but not CU-AP, suggesting an AD pathology-related, hydrophobic functionalization of diffuse plaques facilitating Aß1-40 deposition. Experiments in tgAPPSwe mice verified that, similar to what has been observed in human brain pathology, diffuse deposits display higher levels of Aß1-42 and that Aß plaque maturation over time is associated with increases in Aß1-40. Finally, we found that Aß1-40 deposition is characteristic for cerebral amyloid angiopathy deposition and maturation in both humans and mice. These results indicate that N-terminal Aßx-42 pyroglutamation and Aß1-40 deposition are critical events in priming and maturation of pathogenic Aß from diffuse into cored plaques, underlying neurotoxic plaque development in AD.

GM1 locates to mature amyloid structures implicating a prominent role for glycolipid-protein interactions in Alzheimer pathology.

While the molecular mechanisms underlying Alzheimer's disease (AD) remain largely unknown, abnormal accumulation and deposition of beta amyloid (Aß) peptides into plaques has been proposed as a critical pathological process driving disease progression. Over the last years, neuronal lipid species have been implicated in biological mechanisms underlying amyloid plaque pathology. While these processes comprise genetic features along with lipid signaling as well as direct chemical interaction of lipid species with Aß mono- and oligomers, more efforts are needed to spatially delineate the exact lipid-Aß plaque interactions in the brain. Chemical imaging using mass spectrometry (MS) allows to probe the spatial distribution of lipids and peptides in complex biological tissues comprehensively and at high molecular specificity. As different imaging mass spectrometry (IMS) modalities provide comprehensive molecular and spatial information, we here describe a multimodal ToF-SIMS- and MALDI-based IMS strategy for probing lipid and Aß peptide changes in a transgenic mouse model of AD (tgAPPArcSwe). Both techniques identified a general AD-associated depletion of cortical sulfatides, while multimodal MALDI IMS revealed plaque specific lipid as well as Aß peptide isoforms. In addition, MALDI IMS analysis revealed chemical features associated with morphological heterogeneity of individual Aß deposits. Here, an altered GM1 to GM2/GM3 ganglioside metabolism was observed in the diffuse periphery of plaques but not in the core region. This was accompanied by an enrichment of Aß1-40arc peptide at the core of these deposits. Finally, a localization of arachidonic acid (AA) conjugated phosphatidylinositols (PI) and their corresponding degradation product, lyso-phosphatidylinositols (LPI) to the periphery of Aß plaques was observed, indicating site specific macrophage activation and ganglioside processing.

Molecular imaging mass spectrometry for probing protein dynamics in neurodegenerative disease pathology.

Recent advances in the understanding of basic pathological mechanisms in various neurological diseases depend directly on the development of novel bioanalytical technologies that allow sensitive and specific chemical imaging at high resolution in cells and tissues. Mass spectrometry-based molecular imaging (IMS) has gained increasing popularity in biomedical research for mapping the spatial distribution of molecular species in situ. The technology allows for comprehensive, untargeted delineation of in situ distribution profiles of metabolites, lipids, peptides and proteins. A major advantage of IMS over conventional histochemical techniques is its superior molecular specificity. Imaging mass spectrometry has therefore great potential for probing molecular regulations in CNS-derived tissues and cells for understanding neurodegenerative disease mechanism. The goal of this review is to familiarize the reader with the experimental workflow, instrumental developments and methodological challenges as well as to give a concise overview of the major advances and recent developments and applications of IMS-based protein and peptide profiling with particular focus on neurodegenerative diseases. This article is part of the Special Issue "Proteomics".

Investigating the Role of the Stringent Response in Lipid Modifications during the Stationary Phase in E. coli by Direct Analysis with Time-of-Flight-Secondary Ion Mass Spectrometry.

Escherichia coli is able to rapidly adjust the biophysical properties of its membrane phospholipids to adapt to environmental challenges including starvation stress. These membrane lipid modifications were investigated in glucose starved E. coli cultures and compared to a ΔrelAΔspoT (ppGpp(0)) mutant strain of E. coli, deficient in the stringent response, by means of time-of-flight-secondary ion mass spectrometry (TOF-SIMS). Recent advances in TOF-SIMS, through the implementation of gas cluster ion beams (GCIBs), now permit the analysis of higher mass species from native, underivatized, biological specimen, i.e., intact bacterial cells. Cultures in stationary phase were found to exhibit a radically different lipid composition as compared to cultures in the exponential growth phase. Wild-type E. coli reacted upon carbon starvation by lipid modifications including elongation, cyclopropanation, and increased cardiolipin formation. Observations are consistent with variants of cardiolipins (CL), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), and fatty acids. Notably, despite having a proteomic profile and a gene expression profile somewhat similar to the wild-type during growth, the ppGpp(0) mutant E. coli strain was found to exhibit modified phospholipids corresponding to unsaturated analogues of those found in the wild-type. We concluded that the ppGpp(0) mutant reacts upon starvation stress by elongation and desaturation of fatty acyl chains, implying that only the last step of the lipid modification, the cyclopropanation, is under stringent control. These observations suggest alternative stress response mechanisms and illustrate the role of the RelA and SpoT enzymes in the biosynthetic pathway underlying these lipid modifications.