Unique Patterns of Glycosylation in Plaques and Microglia in Alzheimer’s Disease

Research Background

Alzheimer’s disease (AD) is the most common form of dementia and a devastating neurodegenerative disorder. AD is characterized by two pathological features: extracellular β-amyloid (Aβ) plaques and intracellular phosphorylated Tau neurofibrillary tangles (NFT) inclusions. Dysregulation of microglia is also a key feature in AD pathology. Normally, microglia prune synapses, monitor brain homeostasis threats, and clear cellular debris. However, in AD, microglia respond to pathological aggregates, altering phagocytosis and cytokine secretion, which can have both positive and negative effects on neuropathology.

Research Objective

This study aims to describe the glycosylation landscape of O- and N-linked sialic acid (SA) modifications in the brain tissue of autopsy-confirmed AD patients using a novel combination of histological techniques. Sialic acid-modified glycans play important roles in cell-cell interactions, cell migration, cell adhesion, immune regulation, and membrane excitability. Although previous studies have identified changes in sialylation in AD, the spatial resolution of common methods is limited, and the locations of these sialic acid modifications in cell- or aggregate-associated glycans are still poorly understood.

Research Source

The authors of this paper include Caitlyn Fastenau, Madison Bunce, Mallory Keating, Jessica Wickline, Sarah C. Hopp, and Kevin F. Bieniek, who are affiliated with the Department of Pharmacology, Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, and the Department of Pathology and Laboratory Medicine at the University of Texas Health Science Center at San Antonio. The paper is published in 2024 by the journal “Brain Pathology.”

Research Methods

Acquisition and Processing of Human Brain Tissue

Ten autopsy brains from the Glenn Biggs Institute at the University of Texas Health Science Center at San Antonio were selected for study. These cases were post-mortem confirmed as AD, with composite scores including high AD pathology (n=7), intermediate AD pathology (n=1), and low AD pathology (n=2). All patients exhibited signs of AD during their lifetime.

The left hemisphere was fixed in 10% neutral buffered formalin for at least one month, then coronally sectioned, processed over 28 hours, and embedded in paraffin blocks. Sections included samples from the middle frontal gyrus, hippocampus, and cerebellum, with a thickness of 5μm. Continuous sections and histological staining were then performed.

Immunohistochemistry and Specific Brightfield Staining

A series of histological and immunohistochemical stains were used to visualize Aβ plaques, microglia, and phosphorylated Tau protein, as well as sialic acid modifications. Stains included chromogenic double stains specific for detecting α-2,6 N-sialic acid and O-sialic acid.

Specific steps included deparaffinization, antigen retrieval by steaming, antibody and secondary antibody reactions, chromogen development, Nile blue staining, and coverslip mounting on tissue sections. Quantitative digital pathology techniques were used to assess the quantity and distribution of sialylated glycans in different brain regions.

Data Analysis

Brightfield immunohistochemistry images were obtained using a Leica Aperio AT2 slide scanner and processed using Aperio ImageScope software. These images were compared with control tissue to verify the color reversal algorithm. Quantitative pathology analysis required aligning sequential sections to ensure each region of interest (ROI) was in the same location across all five sections. ROIs were placed in the middle frontal gyrus, hippocampus CA1, and cerebellum molecular layer. When the ROI was located at the site of an Aβ plaque, it was compared to an adjacent area without Aβ pathology (internal control).

Fluorescent Double Staining

To better understand the role of α-2,6 N-sialylation in microglia, co-immunofluorescence staining for CD163, a marker of plaque-associated microglia, was performed, with further analysis by super-resolution imaging techniques.

Main Results

Significant Increase of α-2,6 N-Sialic Acid in Aβ Plaques

The study found that the mean percentage area of α-2,6 N-acid in plaque environments was significantly higher than in non-plaque areas, with significant differences across different brain regions. In the middle frontal gyrus and hippocampus plaque regions, α-2,6 N-sialic acid was significantly higher than in non-plaque regions.

No Significant Differences in O-Sialic Acid

For O-sialic acid neutral and sulfate modifications, although an increase was observed in some subtypes in plaque regions, overall differences were not significant. This suggests a weaker association between O-sialic acid and AD pathology in different brain regions.

α-2,6 N-Sialylation of Microglia

Further analysis revealed that about 65% of microglia in Aβ plaques exhibited a higher level of α-2,6 N-sialylation. Similarly, cases with high AD pathology showed significantly more sialylated microglia than cases with low pathology, especially in plaque regions.

Glycosylation Patterns of Plaques and Diffused Plaques

In the subgroup analysis of core and diffuse plaques, no significant differences were found, but it was suggested that peripherally sialylated microglia around core plaques might play a potential role in plaque compactness.

Sialylation in Tau Pathology

The study also explored the relationship between sialylation and phosphorylated tau pathology, finding that although there was higher phosphorylated tau pathology in the hippocampus CA2 region, the increase in α-2,6 N-sialylation was not significant, and there was no significant positive correlation between α-2,6 N-sialylation and phosphorylated tau.

Important Conclusions and Value

Overall, this study systematically localized and quantified N- and O-linked sialic acid modifications in AD brain tissue using novel histological methods. The study shows that there is a significant increase in N-linked sialylation in microglia and their surrounding pathological environments in AD. This increase may have potential therapeutic target implications for AD protein aggregates and suggests a functional research outlook on α-2,6 N-sialylated microglia in AD pathology.

Research Highlights

1. Novel Research Methods and Techniques: Using advanced histological techniques combined with quantitative digital pathology to precisely locate sialylation.
2. Important Findings: Significant increase in α-2,6 N-sialylation in plaque-associated microglia, indicating their importance in AD pathology.
3. Potential Therapeutic Target: The findings provide new therapeutic strategy directions focusing on microglial glycosylation intervention.

Additional Information

The research was supported by multiple grants from the National Institutes of Health and the Texas Alzheimer’s Research and Care Consortium. All results were based on data from the AGORA platform created by the AMP-AD consortium, supporting AD target discovery. Data can be accessed at DOI:10.57718/agora-adknowledgeportal.

The authors express their heartfelt gratitude to the brain donors and their families from the South Texas Alzheimer’s Disease Research Center at the University of Texas Health Science Center. All datasets used in the article are included in the supplementary data, following the review standards of the ethics committee.

This study provides valuable data for further understanding microglial response strategies and glycosylation modifications in AD and promotes in-depth exploration of potential therapeutic targets for treating AD.