The blood-brain barrier (BBB) is extremely specialized to preserve the hemostatic states and shield the brain from dangerous circulating substances in the blood. The luminal surface of the BBB is coated by the brain endothelium glycocalyx layer, which is a carbohydrate-rich meshwork made predominantly of glycolipids, proteoglycans, and glycoproteins. This lumen mediates various processes, such as transport, cell adhesion, morphology, and signaling. The glycocalyx layer is an essential component of the BBB. Although this is the first barrier layer between the brain vasculature and blood, little is understood about its structure and role in supporting the operation of the BBB, particularly in aging (healthy conditions) and neurodegenerative diseases. This was well demonstrated in a recent study published in Nature.
This study used C57BL/6 mice aged 16-21 months (aged mice) and 3 months (young mice) along with post-mortem fresh frozen human brain tissue diagnosed with Alzheimer’s disease. Various analysis techniques were employed in this study, including transmission electron microscopy (TEM), flow cytometry, immunofluorescence method, BBB leakage assay, liquid chromatography-tandem mass spectrometry (LC-MS-MS), ribonucleic acid (RNA) sequencing glycosylation-related gene analysis, reactive oxygen species (ROS) assay and behavioral tests. All statistical analyses were conducted using GraphPad Prism and R studio (v4 1.1) software.
Aged mice showed a significant decrease in the luminal glycocalyx layers of cortical capillaries compared to young mice. The quantitative analysis revealed that reduction in both average glycocalyx area (0.367±0.054 versus 0.207±0.047 as a proportion of lumen area) and average glycocalyx thickness (0.540±0.086 μm vs. 0.232±0.092 μm) with aging.
The pathway assay identified a significant upregulation of genes associated with heparin sulfate metabolism including syndecan-4 (Sdc4), heparan sulfate glucosamine 3-O-sulfotransferase 1 (Hs3st1), exostosin-like glycosyltransferase 2 (Extl2), and glypican-5 (Gpc5) in aged brain endothelial cells compared to young. In contrast, enzymes included in mucin-type O-glycan biosynthesis such as polypeptide N-acetyl galactosaminyl transferase 10 (Galnt10), beta-1,3-N-acetylglucosaminyltransferase 3 (B3gnt3), polypeptide N-acetylgalactosaminyltransferase 2 (Galnt2), and core 1 β1,3-galactosyltransferase (C1galt1) were downregulated in aged cells compared to young cells.
Fluorescence analysis demonstrated that significant improvements in the gens of chondroitin sulfate, heparan sulfate, and hyaluronan were observed with aging. Also, a significant reduction in mucin-domain glycoproteins genes and no significant alteration in α2,6- or α2,3-linked sialic acids were reported with aging.
Specifically, age-dependent reduction in luminal secreted protease of C1 esterase inhibitor (StcE [E447D]–AF647) signal appeared in the brain. There was no significant difference observed in luminal cerebrovascular labeling (StcE[E447D] and Sambucus nigra agglutinin [SNA], perfusion of Cy3-conjugated SNA [SNA–Cy3]) between aged and young mice.
Administration of Intravenous STcE injection in young mice significantly degraded the glycocalyx layer after 24 hours, disrupted luminal mucin-type O-glycosylation, reduced StcE (E447D)–AF647 labeling, and enhanced BBB permeability. Prolonged treatment caused the BBB dysfunction and cerebral hemorrhaging.
Adeno-associated virus-mediated microRNA targeting C1galt1 (AAV-miR-C1galt1) efficiently decreased the levels of C1galt1 and reduced StcE(E447D)–AF647 labeling compared to AAV-enhanced green fluorescent protein (AAV-EGFP). These reductions were reported in aged mice compared to young mice. The downregulation of C1galt1enzymes in endothelial cells was observed during aging and neurodegenerative diseases, leading to BBS dysfunction.
RNA sequencing analysis demonstrated the downregulation of genes regulating transforming growth factor-beta (TGFβ) signaling, oxidative stress, and vascular integrity in StcE-treated mice compared to saline-treated mice (control). This led to the accumulation of ROS and a reduction in claudin-5 (CLDN5) tight junction protein, ultimately disrupting the BBB integrity.
Moreover, aged mice did not significantly improve behavioral assessments compared to young mice after AAV-C1galt1 treatment. Overexpression of B3gnt3 was found in aged mice. This promoted the restoration of the homeostasis process and increased cognitive functions.
In conclusion, this study highlights the significant effect of glycocalyx dysregulation on brain health and BBB integrity during aging and neurodegenerative diseases. Future research is necessary for understanding the roles of diverse glycans and glycoconjugates in brain function and neurodegeneration.
Reference: Shi SM, Suh RJ, Shon DJ, et al. Glycocalyx dysregulation impairs blood–brain barrier in ageing and disease. Nature. 2025. doi:10.1038/s41586-025-08589-9
