Shedding Light on the Molecular Pathology of Amyloid Plaques in Transgenic Alzheimer’s Disease Mice Using Multimodal MALDI Imaging Mass Spectrometry

Senile plaques formed by aggregated amyloid β peptides are one of the major pathological hallmarks of Alzheimer’s disease (AD) which have been suggested to be the primary influence triggering the AD pathogenesis and the rest of the disease process. However, neurotoxic Aβ aggregation and progression are associated with a wide range of enigmatic biochemical, biophysical and genetic processes. MALDI imaging mass spectrometry (IMS) is a label-free method to elucidate the spatial distribution patterns of intact molecules in biological tissue sections. In this communication, we utilized multimodal MALDI-IMS analysis on 18 month old transgenic AD mice (tgArcSwe) brain tissue sections to enhance molecular information correlated to individual amyloid aggregates on the very same tissue section. Dual polarity MALDI-IMS analysis of lipids on the same pixel points revealed high throughput lipid molecular information including sphingolipids, phospholipids, and lysophospholipids which can be correlated to the ion images of individual amyloid β peptide isoforms at high spatial resolutions (10 μm). Further, multivariate image analysis was applied in order to probe the multimodal MALDI-IMS data in an unbiased way which verified the correlative accumulations of lipid species with dual polarity and Aβ peptides. This was followed by the lipid fragmentation obtained directly on plaque aggregates at higher laser pulse energies which provided tandem MS information useful for structural elucidation of several lipid species. Majority of the amyloid plaque-associated alterations of lipid species are for the first time reported here. The significance of this technique is that it allows correlating the biological discussion of all detected plaque-associated molecules to the very same individual amyloid plaques which can give novel insights into the molecular pathology of even a single amyloid plaque microenvironment in a specific brain region. Therefore, this allowed us to interpret the possible roles of lipids and amyloid peptides in amyloid plaque-associated pathological events such as focal demyelination, autophagic/lysosomal dysfunction, astrogliosis, inflammation, oxidative stress, and cell death

Novel Trimodal MALDI Imaging Mass Spectrometry (IMS3) at 10 μm Reveals Spatial Lipid and Peptide Correlates Implicated in Aβ Plaque Pathology in Alzheimer’s Disease

Multimodal chemical imaging using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) can provide comprehensive molecular information in situ within the same tissue sections. This is of relevance for studying different brain pathologies such as Alzheimer’s disease (AD), where recent data suggest a critical relevance of colocalizing Aβ peptides and neuronal lipids. We here developed a novel trimodal, high-resolution (10 μm) MALDI imaging MS (IMS) paradigm for negative and positive ion mode lipid analysis and subsequent protein ion imaging on the same tissue section. Matrix sublimation of 1,5-diaminonaphthalene (1,5-DAN) enabled dual polarity lipid MALDI IMS on the same pixel points at high spatial resolutions (10 μm) and with high spectral quality. This was followed by 10 μm resolution protein imaging on the same measurement area, which allowed correlation of lipid signals with protein distribution patterns within distinct cerebellar regions in mouse brain. The demonstrated trimodal imaging strategy (IMS3) was further shown to be an efficient approach for simultaneously probing Aβ plaque-associated lipids and Aβ peptides within the hippocampus of 18 month-old transgenic AD mice (tgArcSwe). Here, IMS3 revealed a strong colocalization of distinct lipid species including ceramides, phosphatidylinositols, sulfatides (Cer 18:0, PI 38:4, ST 24:0) and lysophosphatidylcholines (LPC 16:0, LPC 18:0) with plaque-associated Aβ isoforms (Aβ 1–37, Aβ 1–38, Aβ 1–40). This highlights the potential of IMS3 as an alternative, superior approach to consecutively performed immuno-based Aβ staining strategies. Furthermore, the IMS3 workflow allowed for multimodal in situ MS/MS analysis of both lipids and Aβ peptides. Altogether, the here presented IMS3 approach shows great potential for comprehensive, high-resolution molecular analysis of histological features at cellular length scales with high chemical specificity. It therefore represents a powerful approach for probing the complex molecular pathology of, e.g., neurodegenerative diseases that are characterized by neurotoxic protein aggregation

Multimodal Chemical Imaging of Amyloid Plaque Polymorphism Reveals Aβ Aggregation Dependent Anionic Lipid Accumulations and Metabolism

Amyloid plaque formation constitutes one of the main pathological hallmarks of Alzheimer’s disease (AD) and is suggested to be a critical factor driving disease pathogenesis. Interestingly, in patients that display amyloid pathology but remain cognitively normal, Aβ deposits are predominantly of diffuse morphology suggesting that cored plaque formation is primarily associated with cognitive deterioration and AD pathogenesis. Little is known about the molecular mechanism responsible for conversion of monomeric Aβ into neurotoxic aggregates and the predominantly cored deposits observed in AD. The structural diversity among Aβ plaques, including cored/compact- and diffuse, may be linked to their distinct Aβ profile and other chemical species including neuronal lipids. We developed a novel, chemical imaging paradigm combining matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) and fluorescent amyloid staining. This multimodal imaging approach was used to probe the lipid chemistry associated with structural plaque heterogeneity in transgenic AD mice (tgAPP_Swe) and was correlated to Aβ profiles determined by subsequent laser microdissection and immunoprecipitation-mass spectrometry. Multivariate image analysis revealed an inverse localization of ceramides and their matching metabolites to diffuse and cored structures within single plaques, respectively. Moreover, phosphatidylinositols implicated in AD pathogenesis, were found to localize to the diffuse Aβ structures and correlate with Aβ1–42. Further, lysophospholipids implicated in neuroinflammation were increased in all Aβ deposits. The results support previous clinical findings on the importance of lipid disturbances in AD pathophysiology and associated sphingolipid processing. These data highlight the potential of multimodal imaging as a powerful technology to probe neuropathological mechanisms

On-Tissue Chemical Derivatization for Comprehensive Mapping of Brain Carboxyl and Aldehyde Metabolites by MALDI–MS Imaging

The visualization of small metabolites by MALDI mass spectrometry imaging in brain tissue sections is challenging due to low detection sensitivity and high background interference. We present an on-tissue chemical derivatization MALDI mass spectrometry imaging approach for the comprehensive mapping of carboxyls and aldehydes in brain tissue sections. In this approach, the AMPP (1-(4-(aminomethyl)phenyl)pyridin-1-ium chloride) derivatization reagent is used for the covalent charge-tagging of molecules containing carboxylic acid (in the presence of peptide coupling reagents) and aldehydes. This includes free fatty acids and the associated metabolites, fatty aldehydes, dipeptides, neurotoxic reactive aldehydes, amino acids, neurotransmitters and associated metabolites, as well as tricarboxylic acid cycle metabolites. We performed sensitive ultrahigh mass resolution MALDI-MS detection and imaging of various carboxyl- and aldehyde-containing endogenous metabolites simultaneously in rodent brain tissue sections. We verified the AMPP-derivatized metabolites by tandem MS for structural elucidation. This approach allowed us to image numerous aldehydes and carboxyls, including certain metabolites which had been undetectable in brain tissue sections. We also demonstrated the application of on-tissue derivatization to carboxyls and aldehydes in coronal brain tissue sections of a nonhuman primate Parkinson’s disease model. Our methodology provides a powerful tool for the sensitive, simultaneous spatial molecular imaging of numerous aldehydes and carboxylic acids during pathological states, including neurodegeneration, in brain tissue