Exploring the Aβ Plaque microenvironment in Alzheimer's disease model mice by multimodal Lipid-Protein-Histology Imaging on a benchtop mass spectrometer

Significance of the Topic

Alzheimer’s disease is marked by extracellular amyloid-β (Aβ) plaques and complex cellular changes. Mapping the lipid and protein microenvironment of these plaques can reveal novel biomarkers and mechanistic insights, aiding therapeutic development.

Objectives and Study Overview

This work demonstrates a multimodal, multiomic imaging workflow on a benchtop MALDI mass spectrometer (neofleX) to visualize lipids, protein markers, and histology on the same brain section from APP/PS1 Alzheimer’s model mice. Key goals included co‐localizing Aβ plaques with glial markers and identifying plaque-associated lipid alterations.

Methodology and Instrumentation

Sample Preparation and Imaging

• Fresh-frozen APP/PS1 and wild-type mouse brain sections (20 µm) mounted on ITO slides.
• Lipid imaging by MALDI in reflector negative mode (20 µm pixel size), followed by MS/MS fragmentation of selected lipids.
• On the same slide, MALDI HiPLEX-IHC performed in reflector positive mode to detect five protein markers (Aβ1-42, NeuN, Iba-1, GFAP, APP).
• H&E staining completed after MALDI runs to guide anatomical reference.

Instrumentation and Software

• Benchtop neofleX MALDI-TOF/TOF mass spectrometer (Bruker).
• Pneumatic matrix application (HTX Technologies).
• Data processing in SCiLS Lab, SCiLS Scope, and M2aia.

Main Findings and Discussion

Protein and Histology Imaging

• H&E staining delineated hippocampus (HIP) and cortex (CTX).
• NeuN highlighted neuronal layers; Aβ1-42 plaques co-localized nearly completely with microglia marker Iba-1 and astrocyte marker GFAP, with partial overlap with APP.

Lipid Distribution and Fragmentation

• Elevated plaque-associated gangliosides GM2 and GM3.
• Accumulation of lysophosphatidylinositol (LPI 18:0) and phosphatidylinositol (PI 38:4) near plaques and in neuronal layers.
• Depletion of sulfatides in plaque regions compared to white matter.
• On-tissue TOF/TOF confirmed lipid identities by characteristic fragment ions (e.g., GM2 d36:1, PI 38:4).

These multiomic overlays reveal a defined subset of lipids and activated glial cells tightly associated with Aβ deposits, underscoring the value of integrated imaging.

Benefits and Practical Applications of the Method

• Enables simultaneous mapping of lipids, protein markers, and histology on a single tissue section.
• Facilitates discovery of spatially resolved lipid and protein biomarkers in neurodegenerative disease models.
• Utilizes an accessible benchtop MALDI-TOF/TOF platform suitable for research laboratories without requiring large-scale instruments.

Future Trends and Potential Applications

• Expansion to other neurodegenerative and metabolic disease models for holistic tissue analysis.
• Higher multiplexing capacity to include additional protein markers and lipid classes.
• Integration with machine learning for automated biomarker pattern recognition.
• Translation toward clinical tissue profiling and personalized neuropathology.

Conclusion

The study establishes a robust workflow for multimodal, multiomic imaging using a benchtop MALDI instrument, uncovering lipid and protein signatures of Aβ plaques in Alzheimer’s model mice. This approach offers a powerful tool for biomarker discovery and mechanistic studies in neurodegeneration.

References

• Müller E, Enzlein T, Niemeyer D, von Ammon L, Stumpo K, Easterling M, Hopf C. Pharmaceuticals. 2025;18(2):252.