Multimodal Chemical Imaging to probe Alzheimer’s Disease Pathology

Significance of the topic

The global rise in Alzheimer’s disease (AD), affecting over 12% of individuals above 65, underscores the urgency to elucidate its molecular underpinnings. Traditional histochemical and immunological approaches lack the spatial resolution and chemical specificity to fully characterize heterogeneous amyloid aggregates and their microenvironment. Advanced chemical imaging, notably matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) combined with structure-sensitive fluorescent probes, offers an integrated platform to map peptides and lipids in situ. This capability is critical for understanding plaque polymorphism, distinguishing pathogenic signatures, and guiding therapeutic strategies.

Study objectives and overview

This study introduces a multimodal chemical imaging workflow to interrogate amyloid plaque polymorphism in transgenic mouse models and human postmortem brain tissue. Key aims include:

• Delineate spatial distribution of lipids and Aβ peptide species associated with diffuse versus cored plaques.
• Integrate hyperspectral fluorescence imaging with MALDI MSI to correlate amyloid structure and chemical composition.
• Compare plaque signatures in sporadic AD (sAD) and cognitively unaffected amyloid-positive (CU-AP) individuals to identify disease-specific biomarkers.

Methodology and workflow

Tissue sections (12 µm) from tgAPPSWE mice and human sAD/CU-AP cases were cryo-mounted onto membrane slides for laser microdissection and onto conductive slides for MALDI MSI. The workflow comprised:

• Double staining with luminescent conjugated oligothiophenes (q-FTAA and h-FTAA) to reveal plaque polymorphism via hyperspectral fluorescence microscopy.
• Laser microdissection of spectrally defined plaques, formic acid extraction, immunoprecipitation enrichment, and MALDI MS profiling to identify Aβ variants.
• Consecutive-section MALDI MSI to generate spatial maps of Aβ1-40, Aβ1-42, pyroglutamated Aβ species, and lipid markers.
• Multivariate image analysis (PCA, hierarchical clustering) to correlate spectral and mass spectral features with plaque morphotypes.

Used instrumentation

• MALDI TOF mass spectrometer for imaging mass spectrometry.
• Laser microdissection system for in situ plaque isolation.
• Fluorescence microscope with hyperspectral detection for LCO probe imaging.
• Immunoprecipitation setup for selective Aβ peptide enrichment.
• Data analysis software for multivariate statistical and image alignment workflows.

Main results and discussion

• Lipid profiling revealed that short-chain gangliosides and ceramides preferentially localize to diffuse plaques, whereas ceramide-1-phosphate is enriched in cored, mature plaques, implicating lipid metabolism in plaque maturation.
• Hyperspectral LCO imaging distinguished compact fibrillar cores (q-FTAA–positive) from diffuse coronas (h-FTAA–positive), enabling targeted microdissection and peptide analysis.
• MALDI MSI of consecutive sections showed Aβ1-42 is uniformly distributed in diffuse plaques of both sAD and CU-AP, while Aβ1-40 predominantly accumulates in the core of cored plaques in sAD.
• Pyroglutamated N-terminal Aβ3pE-42 and Aβ11pE-42 were abundant in sAD plaques but absent in CU-AP, identifying N-terminal truncation as a disease-specific modification.
• Contrary to prior assumptions, Aβ4-42 emerged as a major non-specific metabolite mimicking Aβ3pE antibody binding, cautioning against misinterpretation of immunostaining data.

Benefits and practical applications of the method

This multimodal imaging strategy offers:

• High spatial and molecular resolution to map peptide and lipid distributions within individual plaques.
• Complementary chemical and structural information by combining MALDI MSI with hyperspectral fluorescence.
• Ability to discern disease-specific posttranslational modifications of Aβ, guiding biomarker discovery.
• Applicability to both animal models and human tissue, bridging preclinical and clinical research.

Future trends and possibilities

Anticipated developments include:

• Integration with higher-resolution MS platforms and ion mobility separation for deeper proteomic and lipidomic coverage.
• Automation of image alignment and analysis pipelines using machine learning to classify plaque types and predict disease stage.
• Extension to other neurodegenerative markers (e.g., tau isoforms) and co-localization studies of protein aggregates.
• Translation into clinical diagnostics by validating chemical signatures in large patient cohorts.

Conclusion

The presented multimodal chemical imaging approach advances our understanding of amyloid plaque heterogeneity by linking structural polymorphism with distinct lipid and peptide chemistries. It challenges prevailing notions of plaque composition, highlights disease-specific modifications, and establishes a versatile platform for biomarker discovery and pathological investigation in Alzheimer’s disease.

Reference

1. Braak H, Braak E. Neuropathological staging of Alzheimer related changes. Acta Neuropathologica. 1991;82:239–259.
2. Murray ME, Dickson DW. Is pathological aging a successful resistance against amyloid-beta or preclinical Alzheimer’s disease? Alzheimer’s Research & Therapy. 2014;6:24.
3. Carlred L et al. Probing Amyloid-β Pathology in transgenic Alzheimer’s disease mice using MALDI Imaging Mass Spectrometry. J Neurochem. 2016;138:469–478.
4. Kaya I et al. Delineating Amyloid Plaque Associated Neuronal Sphingolipids using MALDI Imaging. ACS Chem Neurosci. 2017;8:347–355.
5. Kaya I et al. Trimodal MALDI Imaging at 10 μm Reveals Lipid and Peptide Correlates in Alzheimer’s plaques. ACS Chem Neurosci. 2017;8:2778–2790.
6. Kaya I et al. Histology-Compatible MALDI Imaging of Neuronal Lipids for Immunofluorescent Staining. Anal Chem. 2017;89:4685–4694.
7. Klingstedt T et al. Luminescent conjugated oligothiophenes for fluorescent assignment of protein inclusion bodies. Chembiochem. 2013;14:607–616.
8. Michno W et al. Multimodal Imaging of Amyloid Plaque Polymorphism Reveals Lipid Accumulations. Anal Chem. 2018;90:8130–8138.
9. Michno W et al. Pyroglutamation of amyloid-β followed by Aβ1-40 deposition underlies plaque polymorphism. J Biol Chem. 2019;294:6719–6732.