The blood-brain barrier (BBB) is highly specialized to maintain the hemostatic state and protects the brain from harmful circulating substances in the bloodstream. 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 luminal surface mediated the various essential processes such as transport, cell adhesion, morphology, and signaling. The glycocalyx layer is an essential component of the BBB.  Although it serves as a first barrier between the brain vasculature and circulating blood, the structure and functional role of the BBB, particularly in aging (healthy condition) and neurodegenerative diseases, remain poorly understood. 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) used along with post-mortem fresh frozen human brain tissue diagnosed with Alzheimer’s disease. Various analytical 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 behavioural 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 a significant 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 ageing.
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 improvement in genes of chondroitin sulfate, heparan sulfate, and hyaluronan was observed with ageing. Also, a significant reduction in mucin-domain glycoproteins genes and no significant alteration in α2,6- or α2,3-linked sialic acids were observed with ageing.
Specifically, an 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 labelling (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 labelling, and enhanced BBB permeability. Prolonged treatment resulted in the BBB dysfunction and cerebral hemorrhaging.
Adeno-associated virus-mediated microRNA targeting C1galt1 (AAV-miR-C1galt1) efficiently reduced the levels of C1galt1 and reduced StcE(E447D)–AF647 labelling 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 that downregulation of genes regulates transforming growth factor-beta (TGFβ) signaling, oxidative stress, and vascular integrity in StcE-treated mice compared to saline-treated mice (control). This can lead to the accumulation of ROS and a reduction in claudin-5 (CLDN5) tight junction protein, disrupting the BBB integrity.
Moreover, aged mice did not show significant improvements in behavioural 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 ageing and neurodegenerative diseases. Future research is necessary to understand the roles of diverse glycans and glycoconjugates in brain function and neurodegeneration. Â
Reference: Shi SM, Suh RJ, Shon DJ, et al. Glycocalyx dysregulation impairs the blood-brain barrier in aging and disease. Nature. 2025. doi:10.1038/s41586-025-08589-9
